The Mechanism of Translation

8/8/2019 The Mechanism of Translation

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The Mechanism of Translation

Translation involves three steps:

1. Initiation

2. Elongation

3. Termination

Initiation

Translation begins with the binding of the small ribosomal subunit to a specific sequence on the

mRNA chain. The small subunit binds via complementary base pairing between one of its internal

subunits and the ribosome binding site, a sequence of about ten nucleotides on the mRNA

located anywhere from 5 and 11 nucleotides from the initiating codon, AUG.

Figure %: Initiation
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Once the small subunit has bound, a special tRNA molecule, called N-formyl methionine, or fMet,

recognizes and binds to the initiator codon. Next, the large subunit binds, forming what is known

as the initiation complex. With the formation of the initiation complex, the fMet-tRNA occupies the

P site of the ribosome and the A site is left empty. This entire initiation process is facilitated by

extra proteins, called initiation factors that help with the binding of ribosomal subunits and tRNA

to the mRNA chain.

Elongation

With the formation of the complex containing fMet-tRNA in the peptidyl site, an aminoacyl tRNA

with the complementary anticodon sequence can bind to the mRNA passing through the acceptor

site. This binding is aided by elongation factors that are dependent upon the energy from the

hydrolysis of GTP. Elongation factors go through a cycle to regenerate GTP after its hydrolysis.

Now, with tRNA bearing a chain of amino acids in the p site and tRNA containing a single amino

acid in the A site, the addition of a link to the chain can be made. This addition occurs through the

formation of a peptide bond, the nitrogen-carbon bond that forms between amino acid subunits to

form a polypeptide chain. This bond is catalyzed by the enzyme peptidyl transferase.


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Figure %: Peptide Formation

The peptide bond occurs between the carboxyl group on the lowest link in the peptide chain

located at the p site and the amine group on the amino acid in the A group. As a result, the

peptide chain shifts over to the A site, with the original amino acid on the A site as the lowest link

in the chain. The tRNA in the A site becomes peptidyl RNA, and shifts over to the P site.Meanwhile, the ribosome engages in a process called translocation: spurred by elongation

factors, the ribosome moves three nucleotides in the 3' prime direction along the mRNA. In other

words, the ribosome moves so that a new mRNA codon is accessible in the A site.

Introduction


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Translation is the RNA directed synthesis of polypeptides. This process requires all threeclasses of RNA. Although the chemistry of peptide bond formation is relatively simple, theprocesses leading to the ability to form a peptide bond are exceedingly complex. The template forcorrect addition of individual amino acids is the mRNA, yet both tRNAs and rRNAs are involved inthe process. The tRNAs carry activated amino acids into the ribosome which is composed ofrRNA and ribosomal proteins. The ribosome is associated with the mRNA ensuring correctaccess of activated tRNAs and containing the necessary enzymatic activities to catalyze peptidebond formation.

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Historical Perspectives

Early genetic experiments demonstrated:

1. The co-linearity between the DNA and protein encoded by the DNA. Yanofskyshowed that the order of observed mutations in the E. coli tryptophan synthetasegene was the same as the corresponding amino acid changes in the protein.

2. Crick and Brenner demonstrated, from a large series of double mutants of thebacteriophage T4, that the genetic code is read in a sequential manner starting from afixed point in the gene, the code was most likely a triplet and that all 64 possiblecombinations of the 4 nucleotides code for amino acids, i.e. the code is degeneratesince there are only 20 amino acids.

The above mentioned experiments only indicated deductive correlation's regarding the geneticcode. The precise dictionary of the genetic code was originally determined by the use of in vitrotranslation systems derived from E. colicells. Synthetic polyribonucleotides were added to thesetranslation system along with all twenty amino acids. One amino acid at a time was radiolabeled.The first demonstration of the dictionary of the genetic code was with the use of poly(U). Thissynthetic polyribonucleotide encoded the amino acid phenylalanine, i.e. the resulting polypeptidewas poly(F).

The utilization of a variety of repeating di- tri- and tetra polyribonucleotides established theentire genetic code. These results of these experiments confirmed that some amino acids areencoded for by more than one triplet codon, hence the degeneracy of the genetic code. Theseexperiments also established the identity of translational termination codons.

An additional important point to come from these early experiments was that the 5' end of theRNA corresponded to the amino terminus of the polypeptide. This was important since previouslabeling experiments had demonstrated that the N-terminus is the beginning of the elongatingpolypeptide. Therefore, in vitro translation experiments established that the RNA is read in the 5'to 3' direction.

Crick first postulated that translation of the genetic code would be carried out through

mediation of adapter molecules. Each adapter was postulated to carry a specific amino acid andto recognize the corresponding codon. He suggested that the adapters contain RNA becausecodon recognition could then occur by complementarity to the sequences of the codons in themRNA.

During the course ofin vitro protein synthesis and labeling experiments it was shown that theamino acids became transiently bound to a low molecular weight mass fraction of RNA. Thisfraction of RNAs have been termed transfer RNAs (tRNAs) since they transfer amino acids to the
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elongating polypeptide. These results indicate that accurate translation requires two equallyimportant recognition steps:

1. The correct choice of amino acid needs to be made for attachment to thecorrespondingly correct tRNA.

2. Selection of the correct amino acid-charged tRNA by the mRNA. This process is

facilitated by the ribosomes which we will discuss below.

Summary of Experiments to Determine the Genetic Code

1. The genetic code is read in a sequential manner starting near the 5' end of themRNA. This means that translation proceeds along the mRNA in the 5' > 3'direction which corresponds to the N-terminal to C-terminal direction of the amino acidsequences within proteins.

2. The code is composed of a triplet of nucleotides.

3. That all 64 possible combinations of the 4 nucleotides code for amino acids, i.e. thecode is degenerate since there are only 20 amino acids.

The precise dictionary of the genetic code was determined with the use of in vitro translationsystems and polyribonucleotides. The results of these experiments confirmed that some aminoacids are encoded by more than one triplet codon, hence the degeneracy of the genetic code.These experiments also established the identity of translational termination codons.

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The Genetic Code

Shown below are the triplets that are used for each of the 20 amino acids found in eukaryoticproteins. The row on the left side indicates the first nucleotide of each triplet and the row acrossthe top represents the second nucleotide. The wobble position nucleotides are indicated in blue.

The three stop codons are highlighted in red.

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Characteristics of tRNAs

More than 300 different tRNAs have been sequenced, either directly or from theircorresponding DNA sequences. tRNAs vary in length from 6095 nucleotides (1828 kD). Themajority contain 76 nucleotides. Evidence has shown that the role of tRNAs in translation is tocarry activated amino acids to the elongating polypeptide chain. All tRNAs:

1. Exhibit a cloverleaf-like secondary structure.

2. Have a 5'-terminal phosphate.
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3. Have a 7 bp stem that includes the 5'-terminal nucleotide and may contain non-Watson-Crick base pairs, e.g. GU. This portion of the tRNA is called the acceptorsince the amino acid is carried by the tRNA while attached to the 3'-terminal OHgroup.

4. Have a D loop and a TC loop.

Dihydrouridine (D) Pseudouridine ()

5. Have an anti-codon loop.

6. Terminate at the 3'-end with the sequence 5'CCA3'.

7. Contain 13 invariant positions and 8 semi-variant positions.

8. Contain numerous modified nucleotide bases (see Biochemistry of Nucleic Acidsfor structures of several modified nucleotides in tRNAs).

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Activation of Amino Acids

Activation of amino acids is carried out by a two step process catalyzed by aminoacyl-tRNA

synthetases. Each tRNA, and the amino acid it carries, are recognized by individual aminoacyl-tRNA synthetases. This means there exists at least 20 different aminoacyl-tRNA synthetases,there are actually at least 21 since the initiator met-tRNA of both prokaryotes and eukaryotes isdistinct from non-initiator met-tRNAs.

Activation of amino acids requires energy in the form of ATP and occurs in a two step reactioncatalyzed by the aminoacyl-tRNA synthetases. First the enzyme attaches the amino acid to the -phosphate of ATP with the concomitant release of pyrophosphate. This is termed an aminoacyl-adenylate intermediate. In the second step the enzyme catalyzes transfer of the amino acid toeither the 2' or 3'OH of the ribose portion of the 3'-terminal adenosine residue of the tRNAgenerating the activated aminoacyl-tRNA. Although these reaction are freely reversible, theforward reaction is favored by the coupled hydrolysis of PPi.

Accurate recognition of the correct amino acid as well as the correct tRNA is different for eachaminoacyl-tRNA synthetase. Since the different amino acids have different R groups, the enzymefor each amino acid has a different binding pocket for its specific amino acid. It is not theanticodon that determines the tRNA utilized by the synthetases. Although the exact mechanism isnot known for all synthetases, it is likely to be a combination of the presence of specific modifiedbases and the secondary structure of the tRNA that is correctly recognized by the synthetases.

It is absolutely necessary that the discrimination of correct amino acid and correct tRNA bemade by a given synthetase prior to release of the aminoacyl-tRNA from the enzyme. Once the
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product is released there is no further way to proof-read whether a given tRNA is coupled to itscorresponding tRNA. Erroneous coupling would lead to the wrong amino acid being incorporatedinto the polypeptide since the discrimination of amino acid during protein synthesis comes fromthe recognition of the anticodon of a tRNA by the codon of the mRNA and not by recognition ofthe amino acid. This was demonstrated by reductive desulfuration of cys-tRNA cys with Raneynickel generating ala-tRNAcys. Alanine was then incorporated into an elongating polypeptidewhere cysteine should have been.

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The Wobble Hypothesis

As discussed above, 3 of the possible 64 triplet codons are recognized as translationaltermination codons. The remaining 61 codons might be considered as being recognized byindividual tRNAs. Most cells contain isoaccepting tRNAs, different tRNAs that are specific for thesame amino acid, however, many tRNAs bind to two or three codons specifying their cognateamino acids. As an example yeast tRNAphe has the anticodon 5'GmAA3' and can recognizethe codons 5'UUC3' and 5'UUU3'. It is, therefore, possible for non-Watson-Crick basepairing to occur at the third codon position, i.e. the 3' nucleotide of the mRNA codon and the 5'

nucleotide of the tRNA anticodon. This has phenomenon been termed the wobble hypothesis

Diagram showing the various modified nucleotides of tRNAs that are found in the wobbleposition in the anticodon. The top half shows the wobble nucleotides of the anticodon in blue andthe various nucleotides (in red) of the wobble position of the codon that can be found in non-Watson-Crick base-pairs. The lower panel illustrates the opposite showing the wobblenucleotides of the codon in blue and the associated wobble nucleotides of the anticodon in red.

Now that we have charged aminoacyl-tRNAs and the mRNAs to convert nucleotide sequencesto amino acid sequences we need to bring the two together accurately and efficiently. This is the

job of the ribosomes. Ribosomes are composed of proteins and rRNAs.

All living organisms need to synthesis proteins and all cells of an organism need to synthesizeproteins, therefore, it is not hard to imagine that ribosomes are a major constituent of all cells ofall organisms. The make up of the ribosomes, both rRNA and associated proteins are slightlydifferent between prokaryotes and eukaryotes.

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Order of Events in Translation

The ability to begin to identify the roles of the various ribosomal proteins in the processes ofribosome assembly and translation was aided by the discovery that the ribosomal subunits willself assemble in vitro from their constituent parts.

Following assembly of both the small and large subunits onto the mRNA, and given thepresence of charged tRNAs, protein synthesis can take place. To reiterate the process of proteinsynthesis:

1. Synthesis proceeds from the N-terminus to the C-terminus of the protein.

2. The ribosomes "read" the mRNA in the 5' to 3' direction.

3. Active translation occurs on polyribosomes (also termed polysomes). This meansthat more than one ribosome can be bound to and translate a given mRNA at any onetime.

4. Chain elongation occurs by sequential addition of amino acids to the C-terminalend of the ribosome bound polypeptide.

Translation proceeds in an ordered process. First accurate and efficient initiation occurs, thenchain elongation and finally accurate and efficient termination must occur. All three of theseprocesses require specific proteins, some of which are ribosome associated and some of whichare separate from the ribosome, but may be temporarily associated with it.

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Initiation

Initiation of translation in both prokaryotes and eukaryotes requires a specific initiator tRNA,tRNAmeti, that is used to incorporate the initial methionine residue into all proteins. In E. coliaspecific version of tRNAmeti is required to initiate translation, [tRNA

fmeti]. The methionine attached

to this initiator tRNA is formylated. Formylation requires N10

-formy-THF and is carried out after themethionine is attached to the tRNA. The fmet-tRNAfmeti still recognizes the same codon, AUG, asregular tRNAmet. Although tRNAmeti is specific for initiation in eukaryotes it is not a formylatedtRNAmet.

The initiation of translation requires recognition of an AUG codon. In the polycistronicprokaryotic RNAs this AUG codon is located adjacent to a Shine-Delgarno element in the mRNA.The Shine-Delgarno element is recognized by complimentary sequences in the small subunitrRNA (16S in E. coli). In eukaryotes initiator AUGs are generally, but not always, the firstencountered by the ribosome. A specific sequence context, surrounding the initiator AUG, aidsribosomal discrimination. This context is A/GCC

A/GCCAUGA/G in most mRNAs.


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Initiation factor complex often referredto as eIF-4F composed of 3 primarysubunits: eIF-4E, eIF-4A, eIF-4G andat least 2 additional factors: PABP,Mnk1 (or Mnk2)

mRNA binding to 40Ssubunit, ATPase-dependent RNAhelicase activity, interaction between polyA tail and capstructure

PABP: polyA-binding proteinbinds to the polyA tail of mRNAs and provides a link toeIF-4G

Mnk1 and Mnk2eIF-4E kinases

phosphorylate eIF-4E increasing association with capstructure

eIF-4A ATPase-dependent RNA helicase

eIF-4E (see below)5' cap recognition; frequently found overexpressed inhuman cancers, inhibition of eIF4E is currently a targetfor anti-cancer therapies

4E-BP (also called PHAS) 3 knownforms

when de-phosphorylated 4E-BP binds eIF-4E andrepresses its' activity, phosphorylation of 4E-BP occursin response to many growth stimuli leading to release ofeIF-4E and increased translational initiation

eIF-4Gacts as a scaffold for the assembly of eIF-4E and -4A inthe eIF-4F complex, interaction with PABP allows 5'-endand 3'-ends of mRNAs to interact

eIF-4B stimulates helicase, binds simultaneously with eIF-4F

eIF-5release of eIF-2 and eIF-3, ribosome-dependentGTPase

eIF-6 ribosome subunit antiassociation

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Activities of eIF-3

The eIF-3 complex is composed of 13 different subunits whose sizes, nomenclature and

functions are described in the Table below. The importance of the eIF-3 complex in translationinitiation is demonstrated by the fact that assembly of the eIF-2-GTP-met-tRNAimet (the ternary

complex), binding of the ternary complex and other components of the 43Spre-initiation complex(PIC) to the ribosome 40Ssubunit, recruitment of the mRNA to the 43SPIC, and scanning of themRNA for the initiator AUG codon recognition are all dependent on eIF-3 complex activity.Therefore, primary function of the components of eIF-3 is to act as a scaffold for the assembly ofthe PIC and this assembled complex is referred to as the multi-initiation factor complex (MFC).
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NomenclatureHuman subunit

designationFunction(s)

eIF3A p170binds 40Ssubunit, binds eIF-4B, involved in formation ofMFC, recruitment of mRNA and the ternary complex

eIF3B p116binds 40Ssubunit, involved in formation of MFC,recruitment and scanning of mRNA, recruitment of ternarycomplex

eIF3C p110binds 40Ssubunit, involved in formation of MFC,recruitment and scanning of mRNA, recruitment of ternarycomplex, recognition of the initiator AUG

eIF3D p66

eIF3E p48

eIF3F p47proposed to be the binding site for mTOR and p70S6K(see regulation of eIF-4E activity below)

eIF3G p44 binding of eIF-4B

eIF3H p40

eIF3I p36

eIF3J p35 binds 40Ssubunit, involved in formation of the MFC

eIF3K p28

eIF3L p67

eIF3M GA17

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Specific Steps in Translational Initiation

Initiation of translation requires 4 specific steps:

1. A ribosome must dissociate into its' 40Sand 60Ssubunits.

2. A ternary complex termed the preinitiation complex is formed consisting of theinitiator, GTP, eIF-2 and the 40Ssubunit.

3. The mRNA is bound to the preinitiation complex.
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4. The 60Ssubunit associates with the preinitiation complex to form the 80Sinitiationcomplex.

The initiation factors eIF-1 and eIF-3 bind to the 40S ribosomal subunit favoringantiassociation to the 60Ssubunit. The prevention of subunit reassociation allows the preinitiationcomplex to form.

The first step in the formation of the preinitiation complex is the binding of GTP to eIF-2 toform a binary complex. eIF-2 is composed of three subunits, , and . The binary complex thenbinds to the activated initiator tRNA, met-tRNAmet forming a ternary complex that then binds to the40Ssubunit forming the 43S preinitiation complex. The preinitiation complex is stabilized by theearlier association of eIF-3 and eIF-1 to the 40Ssubunit.

The cap structure of eukaryotic mRNAs is bound by specific eIFs prior to association with thepreinitiation complex. Cap binding is accomplished by the initiation factor eIF-4F. This factor isactually a complex of 3 proteins; eIF-4E, A and G. The protein eIF-4E is a 24 kDa protein whichphysically recognizes and binds to the cap structure. eIF-4A is a 46 kDa protein which binds andhydrolyzes ATP and exhibits RNA helicase activity. Unwinding of mRNA secondary structure isnecessary to allow access of the ribosomal subunits. eIF-4G aids in binding of the mRNA to the

43S preinitiation complex.

Once the mRNA is properly aligned onto the preinitiation complex and the initiator met-tRNAmet

is bound to the initiator AUG codon (a process facilitated by eIF-1) the 60Ssubunit associateswith the complex. The association of the 60Ssubunit requires the activity of eIF-5 which has firstbound to the preinitiation complex. The energy needed to stimulate the formation of the 80 Sinitiation complex comes from the hydrolysis of the GTP bound to eIF-2. The GDP bound form ofeIF-2 then binds to eIF-2B which stimulates the exchange of GTP for GDP on eIF-2. When GTPis exchanged eIF-2B dissociates from eIF-2. This is termed the eIF-2 cycle (see diagram below).This cycle is absolutely required in order for eukaryotic translational initiation to occur. The GTPexchange reaction can be affected by phosphorylation of the -subunit of eIF-2.

At this stage the initiator met-tRNAmet is bound to the mRNA within a site of the ribosome

termed the P-site, for peptide site. The other site within the ribosome to which incoming chargedtRNAs bind is termed the A-site, for amino acid site.


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The eIF-2 cycle involves the regeneration of GTP-bound eIF-2 following the hydrolysis of GTPduring translational initiation. When the 40S preinitiation complex is engaged with the 60Sribosome to form the 80S initiation complex, the GTP bound to eIF-2 is hydrolyzed providingenergy for the process. In order for additional rounds of translational initiation to occur, the GDPbound to eIF-2 must be exchanged for GTP. This is the function of eIF-2B which is also calledguanine nucleotide exchange factor (GEF).

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Elongation

The process of elongation, like that of initiation requires specific non-ribosomal proteins. In E.colithese are EFs and in eEFs. Elongation of polypeptides occurs in a cyclic manner such that atthe end of one complete round of amino acid addition the A site will be empty and ready to acceptthe incoming aminoacyl-tRNA dictated by the next codon of the mRNA. This means that not onlydoes the incoming amino acid need to be attached to the peptide chain but the ribosome mustmove down the mRNA to the next codon. Each incoming aminoacyl-tRNA is brought to theribosome by an eEF-1-GTP complex. When the correct tRNA is deposited into the A site theGTP is hydrolyzed and the eEF-1-GDP complex dissociates. In order for additional translocationevents the GDP must be exchanged for GTP. This is carried out by eEF-1 similarly to the GTPexchange that occurs with eIF-2 catalyzed by eIF-2B.

The peptide attached to the tRNA in the P site is transferred to the amino group at theaminoacyl-tRNA in the A site. This reaction is catalyzed by peptidyltransferase. This process istermed transpeptidation. The elongated peptide now resides on a tRNA in the A site. The A siteneeds to be freed in order to accept the next aminoacyl-tRNA. The process of moving thepeptidyl-tRNA from the A site to the P site is termed, translocation. Translocation is catalyzed byeEF-2 coupled to GTP hydrolysis. In the process of translocation the ribosome is moved alongthe mRNA such that the next codon of the mRNA resides under the A site. Followingtranslocation eEF-2 is released from the ribosome. The cycle can now begin again. The ability ofeEF-2 to carry out translocation is regulated by the state of phosphorylation of the enzyme, whenphosphorylated the enzyme is inhibited. Phosphorylation of eEF-2 is catalyzed by the enzyme


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eEF2 kinase (eEF2K). Regulation of eEF2K activity is normally under the control of insulin andCa2+ fluxes. The Ca2+-mediated effects are the result of calmodulin interaction with eEF2K.Activation of eEF2K in skeletal muscle by Ca2+ is important to reduce consumption of ATP in theprocess of protein synthesis during periods of exertion which will lead to release of intracellularCa2+ stores. eEF2K itself is also regulated by phosphorylation and one of the kinases thatphosphorylates the enzyme is regulated by mTOR (see Regulation of eIF-4E below). In addition,the master metabolic regulatory kinase,AMP-activated protein kinase (AMPK) will phosphorylateand activate eEF2K leading to inhibition of eEF-2 activity.

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Termination

Like initiation and elongation, translational termination requires specific protein factorsidentified as releasing factors, RFs in E. coliand eRFs in eukaryotes. There are 2 RFs in E. coliand one in eukaryotes. The signals for termination are the same in both prokaryotes andeukaryotes. These signals are termination codons present in the mRNA. There are 3 terminationcodons, UAG, UAA and UGA.

In E. coli the termination codons UAA and UAG are recognized by RF-1, whereas RF-2recognizes the termination codons UAA and UGA. The eRF binds to the A site of the ribosome inconjunction with GTP. The binding of eRF to the ribosome stimulates the peptidytransferaseactivity to transfer the peptidyl group to water instead of an aminoacyl-tRNA. The resultinguncharged tRNA left in the P site is expelled with concomitant hydrolysis of GTP. The inactiveribosome then releases its mRNA and the 80S complex dissociates into the 40S and 60Ssubunits ready for another round of translation.

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Selenoproteins

Selenium is a trace element and is found as a component of several prokaryotic andeukaryotic enzymes that are involved in redox reactions. The selenium in these selenoproteins isincorporated as a unique amino acid, selenocysteine, during translation. A particularly importanteukaryotic selenoenzyme is glutathione peroxidase. This enzyme is required during the oxidationofglutathione by hydrogen peroxide (H2O2) and organic hydroperoxides.

Structure of the Selenocysteine Residue

Incorporation of selenocysteine by the translational machinery occurs via an interesting andunique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically
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selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNAinteracts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is broughtinto the ribosome by a dedicated specific elongation factor. An element in the 3' non-translatedregion (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as aselenocysteine codon.

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Regulation of eIF-4E Activity

The cellular levels of eIF-4E are the lowest of all eukaryotic initiation factors which makes thisfactor a prime target for regulation. Indeed, at least 3 distinct mechanisms are known to exist thatregulate the level and activity of eIF-4E. These include regulation of the level of transcription ofthe eIF-4E gene, post-translational modification via phosphorylation and inhibition by interactionwith binding proteins.

Although the exact mechanisms used to upregulate the transcription of the eIF-4E gene arenot yet well understood, it is known that exposure of cells to growth factors as well as activation of

T cells leads to increased expression of eIF-4E. The proto-oncogeneMYC is believed to play arole in the transcriptional activation of eIF-4E as 2 functional MYC-binding sites have been foundin the promoter region of the eIF-4E gene. Of significant note is the finding that cells that arestably over-expressing the MYC gene also have enhanced levels of eIF-4E. Quite strikingly it hasbeen shown that promiscuous elevation in the levels of eIF-4E lead to tumorigenesis placing thistranslation factor in the category of proto-oncogene.

Numerous extracellular stimuli (e.g. insulin, EGF, angiotensin II and gastrin) that exert aportion of their effects at the level of enhanced translation do so by affecting the state of eIF-4Ephosphorylation. However, it should be noted that not all signals that lead to increased eIF-4Ephosphorylation lead to increased rates of translation. Changes in eIF-4E phosphorylationcorrelate well with progression through the cell cycle. In resting (G0) cells eIF-4E phosphorylationis low, it increases during G1 and S phase and then declines again in M phase. Phosphorylation

of eIF-4E occurs at one major site which is Ser209 (in the human and mouse proteins).

The primary signal transduction pathway leading to eIF-4E phosphorylation is that involvingthe RAS gene. Many growth factors stimulate activation of RAS in response to binding theircognate receptors. Subsequently, RAS activation leads to the phosphorylation and activation ofMAP-interacting kinase-1 (Mnk1) which in turn phosphorylates eIF-4E. Although the exact effectof eIF-4E phosphorylation is not clearly defined, it may be necessary to increase affinity of eIF-4Efor the mRNA cap structure and for eIF-4G.

The principal mechanism utilized in the regulation of eIF-4E activity is through its interactionwith a family of binding/repressor proteins termed 4EBPs (4E binding proteins) which are widelydistributed in numerous vertebrate and invertebrate organisms. In mammalian cells 3 related4EBPs have been found where 4EBP1 and 4EBP2 are also identified as PHAS-I and PHAS-II

(PHAS refers to properties ofheat and acid stability).

Binding of 4E-BPs to eIF-4E does not alter the affinity of eIF-4E for the cap structure butprevents the interaction of eIF-4E with eIF-4G which in turn suppresses the formation of the eIF-4F complex (see Table of Initiation Factors above). The ability of 4EBPs to interact with eIF-4E iscontrolled via the phosphorylation of specific Ser and Thr residues in 4EBP. Whenhypophosphorylated, 4EBPs bind with high efficiency to eIF-4E but lose their binding capacitywhen phosphorylated. Numerous growth and signal transduction stimulating effectors lead tophosphorylation of 4E-BPs just as these same responses can lead to phosphorylation of eIF-4E.
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There are several signal transduction pathways whose activations lead to phosphorylation of4E-BPs. These include pathways that lead to activation of phosphatidylinositol 3-kinase (PI3K),the Akt Ser/Thr kinase which is also called protein kinase B (PKB) and the FKBP12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR) family of proteins. Akt wasoriginally identified as a virally encoded oncogene and there are now at least three members ofthe PKB/Akt family identified as Akt1, Akt2, and Akt3. The mammalian TOR proteins arehomologs of the yeast TOR proteins that were identified in a screen for yeast mutants resistant torapamycin. Rapamycin is an immunosuppressant used primarily in the prevention of tissuerejection following organ transplantation. Rapamycin functions within cells by binding theimmunophilin FK506-binding protein 12 (FKBP12). Immunophilins are intracellular proteins thatbinds to immunosuppressive drugs such as FK506 and rapamycin. When rapamycin inhibits thekinase activity of FRAP/mTOR it can no longer phosphorylate 4EB. One of the major effects ofinsulin is increased protein synthesis and this effect is elicited, in part, via activation of mTORfunction. For more information on the regulation of protein synthesis by insulin see the InsulinAction page.

Targets for mTOR regulation of translational initiation and elongation. AMPK = AMP-activatedkinase. TSC1 and TSC2 = Tuberous sclerosis tumor suppressors 1 (hamartin) and 2 (tuberin);Rheb = Ras homolog enriched in brain; PKB/Akt = protein kinase B; 4EBP1 = eIF-4E bindingprotein; p70S6K = 70kDa ribosomal protein S6 kinase, also called S6K; eEF2K = eukaryoticelongation factor 2 kinase.

Regulation of mTOR activity is effected via several mechanisms. Activation of AMPK results inphosphorylation and activation of the TSC1/TSC2 complex which results in inhibition of mTOR.AMPK can also phosphorylate and inhibit mTOR. Conversely, activation of PKB (as in the case of
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insulin receptor activation) leads to activation of mTOR either by inhibition of the TSC1/TSC2complex or by phosphorylation and activation of mTOR directly. Activation of mTOR leads tophosphorylation of p70S6K and 4EBP1. The net effect of phosphorylation of 4EBP1 is that it isreleased from eIF-4E allowing eIF-4E to actively bind eIF-4G and recognize the cap structure ofmRNAs. Activated p70S6K phosphorylates and inhibits eEF2K. If eEF2K does not phosphorylateeEF2 then translation elongation proceeds uninhibited.

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Heme Control of Translation

Regulation of initiation in eukaryotes is effected by phosphorylation of a ser(S) residue in the-subunit of eIF-2. Phosphorylated eIF-2 in the absence of eIF-2B is just as active an initiator asnon-phosphorylated eIF-2. However, when eIF-2 is phosphorylated the GDP-bound complex isstabilized and exchange for GTP is inhibited. The exchange of GDP for GTP is mediated by eIF-2B (also called guanine nucleotide exchange factor, GEF). When eIF-2 is phosphorylated it bindseIF-2B more tightly thus slowing the rate of exchange. It is this inhibited exchange that affects therate of initiation.

The phosphorylation of eIF-2 is the result of an activity called heme-controlled inhibitor (HCI)which functions as diagrammed below. HCI is generated in the absence of heme, a mitochondrialproduct. Removal of phosphate is catalyzed by a specific eIF-2 phosphatase which is unaffectedby heme. The presence of HCI was first seen in in vitro translation system derived from lysates ofreticulocytes. Reticulocytes synthesize almost exclusively hemoglobin at an extremely high rate.In an intact reticulocyte eIF-2 is protected from phosphorylation by a specific 67 kDa protein.


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The regulation of translation by heme controlled inhibitor (HCI). Control of translation by hemeis clinically important only in erythrocytes. Erythrocytes are enucleate and contain primarily globinmRNA. When the level of heme (required for the synthesis of biologically active hemoglobin) islow it would be inefficient for erythrocytes to synthesize globin protein. As the level of heme fallsthe activity of HCI increases. HCI is a kinase which phosphorylates eIF-2. When phosphorylated,eIF-2 still hydrolyzes bound GTP to GDP and still interacts with eIF-2B (GEF). However, the rateof eIF-2B-mediated GTP exchange is greatly reduced. This renders eIF-2 incapable of beingused to form a new ternary initiation complex and translational initiation is reduced. When thelevel of heme again rises the activity of HCI is reduced and translational initiation is once againactive.

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Interferon Control of Translation

Regulation of translation can also be induced in virally infected cells. It would benefit a virallyinfected cell to turn off protein synthesis to prevent propagation of the viruses. This isaccomplished by the induced synthesis of interferons (IFs). There are 3 classes of IFs. Theleukocyte or-IFs, the fibroblast or-IFs and the lymphocyte or-IFs. IFs are induced by


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dsRNAs and themselves induce a specific kinase termed RNA-dependent protein kinase (PKR)that phosphorylates eIF-2 thereby shutting off translation in a similar manner to that of hemecontrol of translation. Additionally, IFs induce the synthesis of 2'-5'-oligoadenylate, pppA(2'p5'A)n,that activates a pre-existing ribonuclease, RNase L. RNase L degrades all classes of mRNAsthereby shutting off translation.

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Iron Control of Translation

Regulation of the translation of certain mRNAs occurs through the action of specific RNA-binding proteins. Protein of this class have been identified that bind to sequences in either the 5'non-translated region (5'-UTR) or 3'-UTR. Two particularly interesting and important regulatoryschemes related to iron metabolism encompass RNA binding proteins that bind to either the 5'-UTR of one mRNA or the 3'-UTR of another.

The transferrin receptor is a protein located in the plasma membrane that binds the proteintransferrin. Transferrin is the major iron transport protein in the plasma. When iron levels are lowthe rate of synthesis of the transferrin receptor mRNA increases so that cells can take up more

iron. This regulation occurs through the action of an iron response element binding protein (IRBP)that binds to specific iron response elements (IREs) in the 3'-UTR of the transferrin receptormRNA. These IREs form hair-pin loop structures that are recognized by IRBP. This IRBP is aniron-deficient form of aconitase, the iron-requiring enzyme of theTCA cycle. When iron levels arelow, IRBP is free of iron and can therefore, interact with the IREs in the 3'-UTR of the transferrinreceptor mRNA. Transferrin receptor mRNA with IRBP bound is stabilized from degradation.Conversely, when iron levels are high, IRBP binds iron then cannot interact with the IREs in thetransferrin receptor mRNA. The effect is an increase in degradation of the transferrin receptormRNA.

A related, but opposite, phenomenon controls the translation of the ferritin mRNA. Ferritin isan iron-binding protein that prevents toxic levels of ionized iron (Fe2+) from building up in cells.The ferritin mRNA has an IRE in its 5'-UTR. As with the transferrin receptor story, when iron

levels are high, IRBP cannot bind to the IRE in the 5'-UTR of the ferritin mRNA. This allows theferritin mRNA to be translated. Conversely, when iron levels are low, the IRBP binds to the IRE inthe ferrritin mRNA preventing its translation.

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Protein Synthesis Inhibitors

Many of the antibiotics utilized for the treatment of bacterial infections as well as certain toxinsfunction through the inhibition of translation. Inhibition can be effected at all stages of translationfrom initiation to elongation to termination.

Several Antibiotic and Toxin inhibitors of Translation

Inhibitor Comments

Chloramphenicol inhibits prokaryotic peptidyl transferase
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Streptomycin inhibits prokaryotic peptide chain initiation, also induces mRNA misreading

Tetracycline

inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

Neomycin similar in activity to streptomycin

Erythromycin inhibits prokaryotic translocation through the ribosome large subunit

Fusidic acidsimilar to erythromycin only by preventing EFG from dissociating from thelarge subunit

Puromycin

resembles an aminoacyl-tRNA, interferes with peptide transfer resulting in

premature termination in both prokaryotes and eukaryotes

Diptheria toxin catalyzes ADP-ribosylation of and inactivation of eEF-2, eEF-2 contains amodified His residue known as dipthamide, it is this resudue that is the targetof diptheria toxin


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ADP-ribosylated dipthamide residue

Ricinfound in castor beans, catalyzes cleavage of the eukaryotic large subunitrRNA

Cycloheximide

inhibits eukaryotic peptidyltransferase


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Figure %: Translocation

With the A site open again, the next appropriate aminoacyl tRNA can bind there and the same

reaction takes place, yielding a three-amino acid peptide chain. This process repeats, creating a

polypeptide chain in the P site of the ribosome. A single ribosome can translate 60 nucleotidesper second. This speed can be vastly augmented when ribosomes link up to form polyribosomes.

Termination

Translation ends when one of three stop codons, UAA, UAG, or UGA, enters the A site of

the ribosome. There are no aminoacyl tRNA molecules that recognize these sequences. Instead,


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release factors bind to the P site, catalyzing the release of the completed polypeptide chain and

separating the ribosome into its original small and large subunits.

Fig. 1: Transfer RNA (tRNA)

Translation of mRNA by tRNA: Formation of the Initiation Complex

Codon Sheet

To initiate translation, a 30S ribosomal subunitbinds to a short nucleotide sequence on the mRNAcalled the ribosome binding site. However, translation doesn't usually begin until the 30Sribosomal subunit reaches the first AUG sequence in the mRNA. For this reason, AUG is knownas the start codon. At this point, an initiation complex composed of the 30S subunit, a tRNAhaving the anticodon UAC and carrying an altered form of the amino acid methionine (N-formylmethionine or f-Met), and proteins called initiation factors is formed.
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Fig. 4: Translation of mRNA by tRNA: 50S Ribosomal Subunit Attaches to the InitiationComplex.

Codon Sheet (Fig. 2)

A50S ribosomal subunit then attaches to the initiation complex and the initiation factors leave.This forms the 70S ribosome.

Fig. 5A: Translation of mRNA by tRNA.


Now an aminoacyl-tRNA with an anticodon complementary to the third codon, GGA, comes intothe "A" site of the ribosome.
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Translation of mRNA by tRNA.


Once the anticodon of the tRNA at the "A" site forms hydrogen bonds with the second codon along the

mRNA, the amino acid being held by the tRNA at the "P" site of the ribosome is enzymatically removed

and forms a peptide bond with the amino acid carried by the tRNA at the "A" site.

Termination:In comparison to initiation and elongation, termination is relatively a simple process.Multiple cycles of elongation occur culminating in polymerization

of the specific amino acids into a protein molecule. There is no tRNA with an anticodon capable of

recognizing such a termination signal.

Releasing factors (eRF) are capable of recognizing termination signal residues in the A site. The releasing

factor, in conjugation with GTP and the peptidyl transferases, promotes the hydrolysis of the bond between

the peptide and the tRNA occupying the P site. The ribosome dissociates into 40S and 60S subunits.

Prokaryotic translation

Initiation
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The process of initiation of translation in prokaryotes.

Initiation of translation in prokaryotes involves the assembly of the components of thetranslation system which are: the two ribosomalsubunits (50S & 30S subunits), the mRNA to betranslated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid), GTP(as a source of energy), and threeinitiation factors(IF1,IF2, andIF3) which help the assembly ofthe initiation complex.[1]

The ribosome has three sites: the A site, the P site, and the E site. The A site is the point ofentry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-tRNAf

Met, which enters atthe P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site whichis the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide

chain.

Elongation

Elongation of thepolypeptide chain involves addition ofamino acids to the carboxyl end of thegrowing chain. The growing proteinexits the ribosomethrough the polypeptide exit tunnel in thelarge subunit[2].

Elongation starts when the fmet-tRNA enters the P site, causing a conformational changewhich opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated byelongation factor-Tu (EF-Tu), a small GTPase. Now the P site contains the beginning of thepeptide chain of the protein to be encoded and the A site has the next amino acid to be added tothe peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from

the tRNA in the P site and a peptide bond is formed between the last amino acids of thepolypeptide and the amino acid still attached to the tRNA in the A site. This process, known as

peptide bond formation, is catalyzed by a ribozyme (the 23S ribosomal RNAin the 50S ribosomalsubunit). Now, the A site has the newly formed peptide, while the P site has an uncharged tRNA(tRNA with no amino acids). In the final stage of elongation, translocation, the ribosome moves 3nucleotides towards the 3'end of mRNA. Since tRNAs are linked to mRNA by codon-anticodonbase-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A siteto the P site and moving the uncharged tRNA to the E exit site. This process is catalyzed byelongation factor G (EF-G).
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The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).

Termination

Termination occurs when one of the three termination codons moves into the A site. These

codons are not recognized by any tRNAs. Instead, they are recognized by proteins called releasefactors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAAand UGA stop codons). These factors trigger the hydrolysis of the esterbond in peptidyl-tRNAand the release of the newly synthesized protein from the ribosome. A third release factor RF-3catalyzes the release of RF-1 and RF-2 at the end of the termination process.

Polysomes

Translation is carried out by more than one ribosome simultaneously. Because of the relativelylarge size of ribosomes, they can only attach to sites on mRNA 35 nucleotides apart. Thecomplex of one mRNA and a number of ribosomes is called apolysomeor polyribosome.

Effect of antibioticSeveral antibiotics exert their action by targeting the translation process

in bacteria. They exploit the differences between prokaryotic and eukaryotictranslation mechanisms to selectively inhibit protein synthesis in bacteria without

affecting the host.

Secreted and Membrane-Associated Proteins

Proteins that are membrane bound or are destined for excretion are synthesized by ribosomesassociated with the membranes of the endoplasmic reticulum (ER). The ER associated withribosomes is termed rough ER (RER). This class of proteins all contain an N-terminus termed asignal sequence or signal peptide. The signal peptide is usually 13-36 predominantlyhydrophobic residues. The signal peptide is recognized by a multi-protein complex termed thesignal recognition particle (SRP). This signal peptide is removed following passage through theendoplasmic reticulum membrane. The removal of the signal peptide is catalyzed by signalpeptidase. Proteins that contain a signal peptide are called preproteins to distinguish them fromproproteins. However, some proteins that are destined for secretion are also further proteolyzedfollowing secretion and, therefore contain pro sequences. This class of proteins is termedpreproproteins.
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Mechanism of synthesis of membrane bound or secreted proteins. Ribosomes engage the ERmembrane through interaction of the signal recognition particle, SRP in the ribosome with theSRP receptor in the ER membrane. As the protein is synthesized the signal sequence is passedthrough the ER membrane into the lumen of the ER. After sufficient synthesis the signal peptideis removed by the action of signal peptidase. Synthesis will continue and if the protein is secretedit will end up completely in the lumen of the ER. If the protein is membrane associated a stoptransfer motif in the protein will stop the transfer of the protein through the ER membrane. Thiswill become the membrane spanning domain of the protein.

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Proteolytic Cleavage

Most proteins undergo proteolytic cleavage following translation. The simplest form of this isthe removal of the initiation methionine. Many proteins are synthesized as inactive precursors thatare activated under proper physiological conditions by limited proteolysis. Pancreatic enzymesand enzymes involved in clotting are examples of the latter. Inactive precursor proteins that areactivated by removal of polypeptides are termed proproteins.

A complex example of post-translational processing of a preproprotein is the cleavage ofprepro-opiomelanocortin (POMC) synthesized in the pituitary (see the Peptide Hormones pagefor discussion of POMC). This preproprotein undergoes complex cleavages, the pathway of whichdiffers depending upon the cellular location of POMC synthesis.

Another is example of a preproprotein is insulin. Since insulin is secreted from the pancreas ithas a prepeptide. Following cleavage of the 24 amino acid signal peptide the protein folds intoproinsulin. Proinsulin is further cleaved yielding active insulin which is composed of two peptidechains linked togehter through disulfide bonds.
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Still other proteins (of the enzyme class) are synthesized as inactive precursors calledzymogens. Zymogens are activated by proteolytic cleavage such as is the situation for severalproteins of the blood clotting cascade.

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Acylation

Many proteins are modified at their N-termini following synthesis. In most cases the initiatormethionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid. Acetyl-CoA is the acetyl donor for these reactions. Some proteins have the 14 carbon myristoyl groupadded to their N-termini. The donor for this modification is myristoyl-CoA. This latter modificationallows association of the modified protein with membranes. The catalytic subunit of cyclicAMP-dependent protein kinase (PKA) is myristoylated.

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Methylation

Post-translational methylation of proteins occurs on nitrogens and oxygens. The activatedmethyl donor is S-adenosylmethionine (SAM). The most common methylations are on the -amine of lysine residues. Methylation of lysine residues in histones in DNA is an importantregulator of chromatin structure and consequently of transcriptional activity. Lysine methylationwas originally thought to be a permanent covalent mark, providing long-term signaling, includingthe histone-dependent mechanism for transcriptional memory. However, recent evidence hasshown that lysine methylation, similar to other covalent modifications, can be transient anddynamically regulated by an opposing de-methylation activity. Recent findings indicate thatmethylation of lysine residues affects gene expression not only at the level of chromatin, but alsoby modifying transcription factors.

Additional nitrogen methylations are found on the imidazole ring of histidine, the guanidino

moiety of arginine and the R-group amides of glutamate and aspartate. Methylation of the oxygenof the R-group carboxylates of gutamate and aspartate also takes place and forms methyl esters.Proteins can also be methylated on the thiol R-group of cysteine.

As indicated below, many proteins are modified at their C-terminus by prenylation near acysteine residue in the consensus CAAX. Following the prenylation reaction the protein is cleavedat the peptide bond of the cysteine and the carboxylate residue is methylated by a prenylatedprotein methyltransferase. One such protein that undergoes this type of modification is the proto-oncogene RAS.

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Phosphorylation

Post-translational phosphorylation is one of the most common protein modifications thatoccurs in animal cells. The vast majority of phosphorylations occur as a mechanism to regulatethe biological activity of a protein and as such are transient. In other words a phosphate (or morethan one in many cases) is added and later removed.

Physiologically relevant examples are the phosphorylations that occur in glycogen synthaseand glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas.
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Phosphorylation of synthase inhibits its activity, whereas, the activity of phosphorylase isincreased. These two events lead to increased hepatic glucose delivery to the blood.

The enzymes that phosphorylate proteins are termed kinases and those that removephosphates are termed phosphatases. Protein kinases catalyze reactions of the following type:

ATP + protein phosphoprotein + ADP

In animal cells serine, threonine and tyrosine are the amino acids subject to phosphorylation.The largest group of kinases are those that phsophorylate either serines or threonines and assuch are termed serine/threonine kinases. The ratio of phosphorylation of the three differentamino acids is approximately 1000/100/1 for serine/threonine/tyrosine.

Although the level of tyrosine phosphorylation is minor, the importance of phosphorylation ofthis amino acid is profound. As an example, the activity of numerous growth factor receptors iscontrolled by tyrosine phosphorylation.

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Sulfation

Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in somesecreted proteins (eg gastrin). The universal sulfate donor is 3'-phosphoadenosyl-5'-phosphosulphate (PAPS).

Since sulfate is added permanently it is necessary for the biological activity and not used as aregulatory modification like that of tyrosine phosphorylation.

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Prenylation

Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbongeranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds derived fromthe cholesterol biosynthetic pathway. The isoprenoid groups are attached to cysteine residues atthe carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence

at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where Cis cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. Inorder for the prenylation reaction to occur the three C-terminal amino acids (AAX) are firstremoved. Following attachment of the prenyl group the carboxylate of the cysteine is methylatedin a reaction utilizing S-adenosylmethionine as the methyl donor.

In addition to numerous prenylated proteins that contain the CAAX consensus, prenylation isknown to occur on proteins of the RAB family of RAS-related G-proteins. There are at least 60proteins in this family that are prenylated at either a CC or CXC element in their C-termini. TheRAB family of proteins are involved in signaling pathways that control intracellular membranetrafficking.

Some of the most important proteins whose functions depend upon prenylation are those thatmodulate immune responses. These include proteins involved in leukocyte motility, activation,and proliferation and endothelial cell immune functions. It is these immune modulatory roles of
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many prenylated proteins that are the basis for a portion of the anti-inflammatory actions of thestatin class of cholesterol synthesis-inhibiting drugs due to a reduction in the synthesis offarnesylpyrophosphate and geranylpyrophosphate and thus reduced extent of inflammatoryevents. Other important examples of prenylated proteins include the oncogenic GTP-binding andhydrolyzing protein RAS and the -subunit of the visual protein transducin, both of which arefarnesylated. In addition, numerous GTP-binding and hydrolyzing proteins (termed G-proteins) ofsignal transductioncascades have -subunits modified by geranylgeranylation.

Genetic code

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A series of codons in part of a mRNA molecule. Each codon consists of three

nucleotides, usually representing a singleamino acid.

The genetic code is the set of rules by which information encoded in geneticmaterial (DNA or mRNA sequences) is translated into proteins (amino acidsequences) by living cells. The code defines a mapping between tri-nucleotide

sequences, called codons, and amino acids. With some exceptions,[1]

a tripletcodon in a nucleic acid sequence specifies a single amino acid. Because the vastmajority ofgenesare encoded with exactly the same code (see the RNA codontable), this particular code is often referred to as the canonical or standardgenetic code, or simply the genetic code, though in fact there are many variantcodes. For example, protein synthesis in human mitochondria relies on a geneticcode that differs from the standard genetic code.
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Not all genetic information is stored using the genetic code. All organisms'DNA contains regulatory sequences, intergenic segments, and chromosomalstructural areas that can contribute greatly to phenotype. Those elementsoperate under sets of rules that are distinct from the codon-to-amino acidparadigm underlying the genetic code.

The genetic code

After the structure of DNA was deciphered by James Watson, Thomas W.Donnellan, Francis Crick, Maurice Wilkins and Rosalind Franklin, serious effortsto understand the nature of the encoding of proteins began. George Gamow

postulated that a three-letter code must be employed to encode the 20 standardamino acids used by living cells to encode proteins, because 3 is the smallestinteger n such that 4n is at least 20.[2]

The fact that codons consist of three DNA bases was first demonstrated in theCrick, Brenner et al. experiment. The first elucidation of a codon was done byMarshall Nirenbergand Heinrich J. Matthaei in 1961 at the National Institutes ofHealth. They used a cell-free system to translate a poly-uracil RNA sequence(i.e., UUUUU...) and discovered that the polypeptide that they had synthesizedconsisted of only the amino acid phenylalanine. They thereby deduced that thecodon UUU specified the amino acid phenylalanine. This was followed by

experiments in the laboratory of Severo Ochoa demonstrating that the poly-adenine RNA sequence (AAAAA...) coded for the polypeptide, poly-lysine.[3] andthe poly-cytosine RNA sequence (CCCCC...) coded for the polypeptide, poly-proline.[4] Therefore the codon AAA specified the amino acid lysine, and thecodon CCC specified the amino acid proline. Using different copolymers most ofthe remaining codons were then determined. Extending this work, Nirenberg andPhilip Leder revealed the triplet nature of the genetic code and allowed thecodons of the standard genetic code to be deciphered. In these experiments
http://en.wikipedia.org/wiki/Phenotypehttp://en.wikipedia.org/wiki/James_D._Watsonhttp://en.wikipedia.org/wiki/James_D._Watsonhttp://en.wikipedia.org/w/index.php?title=Thomas_W._Donnellan&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Thomas_W._Donnellan&action=edit&redlink=1http://en.wikipedia.org/wiki/Francis_Crickhttp://en.wikipedia.org/wiki/Maurice_Wilkinshttp://en.wikipedia.org/wiki/Rosalind_Franklinhttp://en.wikipedia.org/wiki/George_Gamowhttp://en.wikipedia.org/wiki/Genetic_code#cite_note-isbn0-465-09138-5-1%23cite_note-isbn0-465-09138-5-1http://en.wikipedia.org/wiki/Crick,_Brenner_et_al._experimenthttp://en.wikipedia.org/wiki/Marshall_Nirenberghttp://en.wikipedia.org/wiki/Marshall_Nirenberghttp://en.wikipedia.org/wiki/Heinrich_J._Matthaeihttp://en.wikipedia.org/wiki/National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/Cell-free_systemhttp://en.wikipedia.org/wiki/Translation_(biology)http://en.wikipedia.org/wiki/Polypeptidehttp://en.wikipedia.org/wiki/Phenylalaninehttp://en.wikipedia.org/wiki/Severo_Ochoahttp://en.wikipedia.org/wiki/Severo_Ochoahttp://en.wikipedia.org/wiki/Genetic_code#cite_note-pmid13946552-2%23cite_note-pmid13946552-2http://en.wikipedia.org/wiki/Genetic_code#cite_note-pmid13998282-3%23cite_note-pmid13998282-3http://en.wikipedia.org/wiki/Philip_Lederhttp://en.wikipedia.org/wiki/File:GeneticCode21-version-2.svghttp://en.wikipedia.org/wiki/File:GeneticCode21-version-2.svghttp://en.wikipedia.org/wiki/Phenotypehttp://en.wikipedia.org/wiki/James_D._Watsonhttp://en.wikipedia.org/w/index.php?title=Thomas_W._Donnellan&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Thomas_W._Donnellan&action=edit&redlink=1http://en.wikipedia.org/wiki/Francis_Crickhttp://en.wikipedia.org/wiki/Maurice_Wilkinshttp://en.wikipedia.org/wiki/Rosalind_Franklinhttp://en.wikipedia.org/wiki/George_Gamowhttp://en.wikipedia.org/wiki/Genetic_code#cite_note-isbn0-465-09138-5-1%23cite_note-isbn0-465-09138-5-1http://en.wikipedia.org/wiki/Crick,_Brenner_et_al._experimenthttp://en.wikipedia.org/wiki/Marshall_Nirenberghttp://en.wikipedia.org/wiki/Heinrich_J._Matthaeihttp://en.wikipedia.org/wiki/National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/Cell-free_systemhttp://en.wikipedia.org/wiki/Translation_(biology)http://en.wikipedia.org/wiki/Polypeptidehttp://en.wikipedia.org/wiki/Phenylalaninehttp://en.wikipedia.org/wiki/Severo_Ochoahttp://en.wikipedia.org/wiki/Genetic_code#cite_note-pmid13946552-2%23cite_note-pmid13946552-2http://en.wikipedia.org/wiki/Genetic_code#cite_note-pmid13998282-3%23cite_note-pmid13998282-3http://en.wikipedia.org/wiki/Philip_Leder


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various combinations of mRNA were passed through a filter which containedribosomes, the components of cells that translate RNA into protein. Uniquetriplets promoted the binding of specific tRNAs to the ribosome. Leder andNirenberg were able to determine the sequences of 54 out of 64 codons in theirexperiments.[5]

Subsequent work by Har Gobind Khorana identified the rest of the geneticcode. Shortly thereafter, Robert W. Holley determined the structure of transferRNA (tRNA), the adapter molecule that facilitates the process of translating RNAinto protein. This work was based upon earlier studies by Severo Ochoa, whoreceived the Nobel prize in 1959 for his work on the enzymology of RNAsynthesis.[6] In 1968, Khorana, Holley and Nirenberg received the Nobel Prize inPhysiology or Medicine for their work.[7]

Transfer of information via the genetic code

The genome of an organism is inscribed in DNA, or in the case of someviruses, RNA. The portion of the genome that codes for a protein or an RNA isreferred to as a gene. Those genes that code for proteins are composed of tri-nucleotide units called codons, each coding for a single amino acid. Eachnucleotide sub-unit consists of a phosphate, deoxyribose sugar and one of the 4nitrogenous nucleobases. The purine bases adenine (A) and guanine (G) arelarger and consist of two aromatic rings. The pyrimidine bases cytosine (C) andthymine (T) are smaller and consist of only one aromatic ring. In the double-helixconfiguration, two strands of DNA are joined to each other by hydrogen bonds inan arrangement known as base pairing. These bonds almost always formbetween an adenine base on one strand and a thymine on the other strand and

between a cytosine base on one strand and a guanine base on the other. Thismeans that the number of A and T residues will be the same in a given doublehelix, as will the number of G and C residues. [8]:102117 In RNA, thymine (T) isreplaced by uracil (U), and the deoxyribose is substituted by ribose.[8]:127

Each protein-coding gene is transcribed into a template molecule of therelated polymer RNA, known as messenger RNA or mRNA. This, in turn, istranslated on the ribosome into an amino acid chain orpolypeptide.[8]:Chp 12 Theprocess of translation requires transfer RNAs specific for individual amino acidswith the amino acids covalently attached to them, guanosine triphosphate as anenergy source, and a number of translation factors. tRNAs have anticodons

complementary to the codons in mRNA and can be "charged" covalently withamino acids at their 3' terminal CCA ends. Individual tRNAs are charged withspecific amino acids by enzymes known as aminoacyl tRNA synthetases, whichhave high specificity for both their cognate amino acids and tRNAs. The highspecificity of these enzymes is a major reason why the fidelity of proteintranslation is maintained.[8]:464469
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There are 4 = 64 different codon combinations possible with a triplet codon ofthree nucleotides; all 64 codons are assigned for either amino acids or stopsignals during translation. If, for example, an RNA sequence, UUUAAACCC isconsidered and the reading frame starts with the first U (by convention,5' to 3'),there are three codons, namely, UUU, AAA and CCC, each of which specifies

one amino acid. This RNA sequence will be translated into an amino acidsequence, three amino acids long.[8]:521539 A comparison may be made withcomputer science, where the codon is similar to a word, which is the standard"chunk" for handling data (like one amino acid of a protein), and a nucleotide issimilar to a bit, in that it is the smallest unit.

The standard genetic code is shown in the following tables. Table 1 showswhat amino acid each of the 64 codons specifies. Table 2 shows what codonsspecify each of the 20 standard amino acids involved in translation. These arecalled forward and reverse codon tables, respectively. For example, the codonAAU represents the amino acid asparagine, and UGU and UGC represent

cysteine (standard three-letter designations, Asn and Cys, respectively).

[8]:522

RNA codon table

nonpolar polar basic acidic (stop codon)

2nd base

U C A G

1st

base

U

UUU(Phe/F)

PhenylalanineUCU

(Ser/S)

SerineUAU

(Tyr/Y)

TyrosineUGU

(Cys/C)

Cysteine

UUC(Phe/F)

Phenylalanine UCC(Ser/S)

Serine UAC(Tyr/Y)

Tyrosine UGC(Cys/C)

Cysteine

UUA(Leu/L)

LeucineUCA

(Ser/S)

SerineUAA Ochre (Stop) UGA Opal (Stop)

UUG(Leu/L)Leucine

UCG(Ser/S)Serine

UAGAmber(Stop)

UGG(Trp/W)Tryptophan

C

CUU(Leu/L)

LeucineCCU

(Pro/P)

ProlineCAU

(His/H)

HistidineCGU

(Arg/R)

Arginine

CUC(Leu/L)Leucine

CCC(Pro/P)Proline

CAC(His/H)Histidine

CGC(Arg/R)Arginine

CUA(Leu/L)

Leucine

CCA(Pro/P)

Proline

CAA(Gln/Q)

Glutamine

CGA(Arg/R)

Arginine

CUG(Leu/L)Leucine

CCG(Pro/P)Proline

CAG(Gln/Q)Glutamine

CGG(Arg/R)Arginine

AAUU

(Ile/I)

IsoleucineACU

(Thr/T)

ThreonineAAU

(Asn/N)

AsparagineAGU

(Ser/S)

Serine

AUC(Ile/I)

IsoleucineACC

(Thr/T)

ThreonineAAC

(Asn/N)

AsparagineAGC

(Ser/S)

Serine

AUA (Ile/I) ACA (Thr/T) AAA (Lys/K) AGA (Arg/R)
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Isoleucine Threonine Lysine Arginine

AUG[A](Met/M)

MethionineACG

(Thr/T)

ThreonineAAG

(Lys/K)

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The Mechanism of Translation

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