Protein Synthesis Biology 12

Mr. McIntyre

Translation: From messenger RNA to protein:

The information encoded in the DNA is transferred to messenger RNA and then decoded by the ribosome toproduce proteins.

5’-ATGCCTAGGTACCTATGA-3’3’-TACGGATCCATGGATACT-5’

5’-AUGCCUAGGUACCUAUGA-3’

5’-AUG CCU AGG UAC CUA UGA-3’

N-MET-PRO-ARG-TYR-LEU-C

DNA

Transcription

decoded as

Translation

mRNA

Protein

Alanine tRNA

Generalized tRNA

= UH2

Modified BasesFound in tRNAs

tRNAs are activated by amino-acyl tRNA synthetases

Structure of an amino acyl-tRNA synthetase bound to a tRNA

One mechanism for maintaining high fidelity of proteinsynthesis is the high fidelity of aa-tRNA synthetases

Amino-acyl tRNA synthetases:

One synthetase for each amino acida single synthetase may recognize multiple tRNAsfor the same amino acid

Two classes of synthetase. Different 3-dimensional structuresDiffer in which side of the tRNA they recognize and how they bind ATP

Class I - monomeric, acylates the 2’OH on the terminal riboseArg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val

Class II - dimeric, acylate the 3’OH on the terminal riboseAla, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

Two levels of control to ensure that the proper amino acidis incorporated into protein: 1) Charging of the proper tRNA

2) Matching the cognate tRNA to the messenger RNA

Incorporation of amino acids into polypeptide chains I

Incorporation of amino acids into polypeptide chains II

Protein synthesis occurs on ribosomes

Protein synthesis occurs on ribosomes

and mitochondria

Ribosome Assembly

The proteins of each ribosomal subunitare organized aroundrRNA molecules

16S rRNA

Ribosome Assembly: takes place largely in a specialized domain ofthe nucleus, the nucleolus

In the nucleolus, RNA polymerase I transcribes the rDNA repeatsto produce a 45S RNA precursor

The 45S precursor is processedand cleaved intomature rRNAs andribosomal proteinsthen bind to generatethe large and smallribosomal subunits

23S rRNA secondary structure

3D organization of the eukaryotic large subunit rRNA

Ribosomal Proteins decorate the surface of the ribosome

Large subunit. Grey = rRNA Gold = ribosomal proteins

Ribosomal proteins often have extensions that snake into the core of the rRNA structure

Crystal structure of L19 L15 (yellow) positioned in a fragmentof the rRNA (red)

The ribosomal proteins are important for maintaining the stability and integrity of the ribosome, but NOT for catalysis

ie. the ribosomal RNA acts as a ribozyme

Mitochondrialor Prokaryotic

Eukaryotic 60S subunit 80S ribosome 40S subunit

The large and small subunits come together to form the ribosome

The association of the large and small subunits creates the structural features on the ribosome that are essential for protein synthesis

Three tRNA bindingsites:A site = amino-acyltRNA binding site

P site = peptidyl-tRNAbinding site

E site = exit site

In addition to the APE sites there is an mRNA binding groovethat holds onto the message being translated

There is a tunnel through the large subunit that allows thegrowing polypeptide chain to pass out of the ribosome

Peptide bond formation is catalyzed by the large subunit rRNA

Peptide bond formation is catalyzed by the large subunit rRNA

Incorporation of the correct amino acyl-tRNA is determinedby base-pairing interactions between the anticodon of the tRNA and the messenger RNA

Proper reading of theanticodon is the secondimportant quality controlstep ensuring accurateprotein synthesis

=EF-1

Elongation factors Introduce a two-step“Kinetic proofreading”

A second elongation factorEF-G or EF-2, drives the translocation of the ribosome along the mRNA

Together GTP hydrolysisby EF-1 and EF-2 help driveprotein synthesis forward

Termination of translationis triggered by stop codons

Release factor entersthe A site and triggershydrolysis the peptidyl-tRNAbond leading to release of the protein.

Release of the protein causesthe disassociation of the ribosome into its constituentsubunits.

Release Factor is a molecular mimic of a tRNA

eRF1 tRNA

Initiation of Translation

Initiation is controlled differently in prokaryotic and eukaryotic ribosomes

In prokaryotes a single transcript can give rise to multiple proteins

In prokaryotes, specific sequences in the mRNA aroundthe AUG codon, calledShine-Delgarno sequences,are recognized by an intiationcomplex consisting of a Metamino-acyl tRNA, Initiation Factors (IFs) and the smallribosomal subunit

GTP hydrolysis by IF2 coincident with release of the IFs and binding of the largeribosomal subunit leads to formation of a completeribosome,on the mRNAand ready to translate.

Eukaryotic mRNAs have a distinct structure at the 5’ end

Structure of the 7-methyl guanosine cap

The 7me-G cap is requiredfor an mRNA to be translated

In contrast, Eukaryotesuse a scanning mechanismto intiate translation.

Recognition of the AUGtriggers GTP hydrolysisby eIF-2

GTP hydrolysis byeIF2 is a signal forbinding of the largesubunit and beginningof translation

Messenger RNAs are translated on polyribosomes

Protein synthesis is often regulated at the level of translation initiation

An example of control of specific mRNAs: regulation by iron (Fe):

Ferritin is a cytosolic iron binding protein expressed wheniron is abundant in the cell.

Transferrin receptor is a plasma membrane receptor important for the import of iron into the cytosol.

They are coordinately regulated, in opposite directions, bycontrol of protein synthesis.

Regulation by iron (Fe):

There is also general control of translational initiation.

ie. all transcripts of the cell are effected (though the relativeeffect differs between specific mRNAs)

Global downregulation or upregulation can occur in response to various stimuli the most common are

1) Nutrient availabilitylow nutrient (amino acids/carbohydrate) downregulates translation

2) Growth factor signals.stimulation of cell division upregulates translation

General control of translational initiation is exerted throughtwo primary mechanisms.

Control of the phosphorylation of eIF2

Control of the phosphorylation of eIF4 binding proteins

Control of translation by eIF2 phosphorylation

Stimulated byAmino acid deprivation

Control of translation by eIF4E availability

The 7MEG cap binding subunit of eIF4, eIF4E, is sequesteredby eIF4E binding protiens (4E-BPs). The binding of theseproteins is regulated by their phosphorylation state

GrowthFactors

NutrientLimitation

Nutritional signals can control both the recognition of the mRNAand loading of the 40S subunit.

Nutritionalcontrols

2

Modification of the translation machinery is a commonfeature of viral life cycles

e.g. PicornavirusesPolio virusEncephalomyocarditis virus

Picornaviruses have single stranded RNA genomes.

Poliovirus Life Cycle

The poliovirus genome is translated into a single,large polyprotein that then auto-proteolyzes itself intosmaller proteins.

One of these proteins, viral protease 2A cleavesthe translation initiation factor eIF4G so that it can no longer function as a bridge between themethyl cap binding subunit and the 40S subunit

The consequence of this cleavage is that translation of cellular mRNAs stops

But…the viral RNA is still translated due to the presence ofan internal ribosomal entry site (IRES). This acts like a bacterial initiation site to allow Cap-independent initiationfrom internal AUG codons.

What is X?

“X” is not a protein, as suggestedby the textbook model at right,rather it is a structure in the mRNAitself that can bind to the remainingfragment of eIF4G

Some cellular mRNAs are also translated using IRESs

During G2/M phase of the cell cycle, translation is generallydownregulated by activation of 4E-BPs. Many proteins expressedduring this period bypass this control by using IRES elements

Ribosomal Frameshifting

Because translationuses a triplet code,there are three potentialreading frames in each mRNA

As the ribosome translocates, it moves in three nucleotidesteps, ensuring that the frame defined by the AUG is usedthroughout translation

If the ribosome moves 1 or 2 (or 4 or 5) nucleotidesthis produces a frameshift

Many retroviruses induce ribosomal frameshifting in the synthesis of viral proteins

e.g. HIV

Translation Inhibitors are important antibiotics