Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: engelber@cc.huji.ac.il

transcript

Dudi Engelberg

Room 1-517Tel: 658 4718e-mail: engelber@cc.huji.ac.il

The central dogmaof molecular biology

Protein

Transcription

Translation

4Could proteins multiply ?

What do we have RNA for?

Same DNA content in all cells of themulicellular organism?

What is the function of DNA?

Can cells function without DNA?

Are these all nucleotides that appear in DNA and RNA?11

What are the cellular functions of nucleotides?

Some cellular functions of nucleotides1. Building blocks of nucleic acids.

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes.

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions. Involved in many more pottranslational modifications.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).15

Some cellular functions of deoxynucleotides

1. Building blocks of nucleic acids (DNA).

Some cellular functions of deoxynucleotides1. Building blocks of nucleic acids (DNA).

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes (GTP).

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).17

Some deviations from the averaged Watson & Crick model

The pitch angle between base pairs could be 28o - 42o.

Bases could propel (deviate from planarity).

Damages: kinks and covalent bonding inside the helix (usuallyBetween bases).

Presence of unusual bases (in tRNA for example) allows unusualbase pairing and novel structural motifs.

Presence of specific sequences (stretch of purines,palindromes, sequence repeats).

The driving force towardssynthesis is the breakdown ofPPi.

Phosphodiester bond

Mechanism of the basic synthesis reaction of nucleic acids

Addition of nucleotide involves an attack by the 3’-hydroxyl group at the end of the growing RNA molecule on the a phosphate of the oncoming NTP.

Two Mg2+ ions coordinated to the phosphate groups of the NTP and to three Asp residues of the subunit of E. coli RNA polymerase (conserved in most RNA polymerasess in nature).

One Mg2+ ion facilitate the attack by the 3’-hydroxyl group on the a phosphate and the other ion facilitates the displacement of pyrophosphate.

The Mg2+ ions stabilize in fact the transition (intermediate) state.

Polymerization of nucleotides - DNA and RNA biosynthesis1. The reaction is directional; proceeds from 5’end to 3’end. As a result the product is asymetric (5’end different than 3’end.2. The nucleotides (of the same strand) are always linked in a phospho-di-ester bond (a covalent bond).3. Energy is wasted in addition of each monomer. The driving force towards synthesis is degradation of pyrophosphate.4. The precursors are always nucleotides tri-phosphates (NTPs or dNTPs).6. The reaction is directed by a pre exist plan (a template).

(No polymerase is capable of adding nucleotides randomly).

May be there are some - quite important34

Basic characteristics of DNA Pol1. Is not capable of de novo synthesis. Requires: A. A template (as any other polymerase). B. A primer (RNA oligo, nicked DNA, protein?)

2. Possesses two catalytic activities: A. A 5’ to 3’ polymerase activity. B. A 3’ to 5’ exonuclease actiivty.

3. Substrates are only dNTPs.

How DNA Pol is regulated?Does it possess regulatorysites?

DNA replication is semi-conservative

DNA replication is bi-directional

Schematic structure of E. coli replication origin (OriC)245 bp.3 repeats of 13 bp sequences + 4 repeats of 9 bp sequence.These elements are highly conserved in replicationorigins of bacteria.

Initiation step: “opening” DNA “preparing the template beforeany DNA synthesis occurs.

First key step in replication: binding of DnaA protein molecules to the four 9 bp repeats.DnaA binding requires ATP and HU

Second step: binding of DnaB (hexamerix helicase). Two hexamers bind to unwind DNA at two points creating two potential replicating forks.

Third step: binding of SSBs (essential for stabilizing single strand throughout thereplication process) and DNA gyrase (DNA topoII) - this step allows DnaBhelicase to unwind thousands of base-pairs.

DnaA binds cooperatively to form a corearound which OriC DNA is wrapped.At the presence of ATP DnaA melts theDNA of the A-T rich 13 bp tandem repeats.

DnaA molecules recruit two DnaB-DnaCcomplexes, one for each replication forks. (6 DnaC monomers bind the DnaB hexamer.)

Gyrase must be present to relieve topologicalStress - otherwise helicase cannot furthercatalyze unwinding.

Altogether a pre-priming complex is formed:480 kD, 6 nm radius.

Initiation step has prepared the template.

Moving to elongation step:Priming is required.A mechanism for bi-directionality is required.

Leading strand synthesis begins with The synthesis of a short primer (10-60 n)catalyzed by primase (DnaG - special RNA Pol).

Both strands are sybthesized by DNA Pol3.Lagging strand:A new primer is synthesized nearthe replication fork.Synthesis continues until theFragment extends as far as the primerof the previous fragment.

Specific structural capabilities ofDNA Pol 3.

DnaB (helicase) + DnaG (primase) form a functional unit within the replication fork, called primosome.

DNA pol3 - a dimer - one set of subunits synthesize the leading strand and other set the lagging strand.

Once DNA is unwound by DnaB, DnaG associates occasionally with DnaB and synthesizes a short RNA primer.

A new sliding clamp is then positioned at the primer by theclamp-loading complex of Pol 3.

When a synthesis of a fragment is completed, replication halts and the core subunits of Pol 3 dissociate from their sliding clamp and from the new fragment.

subunits on DNA

Exonuclease activity is locatedahead of pol activity

Sequence of the RNA is identical to that of the coding strand(with the replacements of Us for Ts).

Products of the transcription reaction (primary transcript):In prokaryotes: an unstable RNA- rapidly degraded (mRNAor cleaved to give mature products (rRNA, tRNA).

In eukaryotes: modified at the ends (mRNA) and/or cleavedto give mature products (all RNAs).

With the exception of the RNA genomesof certain viruses, all RNA molecules innature (mRNA, tRNA, rRNA, miRNA, snRNA) are derived from information stored in DNA and obtained via transcription.

Namely, just like DNA during replication, RNA is synthesized on DNA template (DNA-dependent RNA synthesis).

Transcription=DNA-dependent RNA synthesis

Polymerization of nucleotides - DNA and RNA biosynthesis1. The reaction is directional; proceeds from 5’end to 3’end. As a result the product is asymetric (5’end different than 3’end.2. The nucleotides (of the same strand) are always linked in a phospho-di-ester bond (a saturated covalent bond).3. Energy is consumed during addition of each monomer. The driving force towards synthesis is degradation of pyrophosphate.4. The precursors are always nucleotides tri-phosphates (NTPs or dNTPs).6. The reaction is directed by a pre exist plan (a template). No plymerase is capable of adding nucleotides randomly.

At its basic enzymatic level, transcription is areaction highly similar to replication

Comparison of replication to transcription (some aspects)

Replication TranscriptionQuantity: The whole genome Parts of the genome

Timing: One time per life cycle some parts - all life time (time is determined by the some parts - some time checkpoint system) some parts - never

Location: From origin to end Many starts and many stops (starts and stops must be most accurate)

DNA substrate: The two strands One strand (could be a different for each particular case

Nucleotidesubstrates: dNTPs NTPs

Comparison of replication to transcription (some aspects)

Replication TranscriptionProofreading: Always Never

Post-reaction repair: Always Never

Fate of product: Remains attached to Released from template template

Processivity: High or low High (from start to termination)

Ligating fragments: Yes No - products are

independent molecules

Sequence of the RNA is identical to that of the coding strand(with the replacements of Us for Ts).

Products of the transcription reaction (primary transcript):In prokaryotes: an unstable RNA- rapidly degraded (mRNAor cleaved to give mature products (rRNA, tRNA).

In eukaryotes: modified at the ends (mRNA) and/or cleavedto give mature products (all RNAs).

RNA Polymerase - general properties

1. Properties similar to DNA Polymerases:- Basic chemical mechanisms: addition of ribonucleotides to the 3’-OH of thechain. Consequently determination of a 5’ to 3’ directionality.- Requires a template.- Adding nucleotides on the basis of optimal hydrogen bonds with the templatestrand (A-U, C-G).

2. Properties specific to RNA Pol- Using only one strand as a template (must make a choice).- Does not require a primer (pppN 5’ end).- Very complex regulation for “choosing” the starting points (which may be different in every cell, in every developmental stage and in ageing.- Does not have a 3’ 5’ exonuclease activity.- The rate of mistake in high (1/104-105).

During a successful round: RNA Pol associates with the starting point and dissociates at the termination point, defining a transcription unit. A transcription unit may include more than one gene

Nomenclature: Upstream. Downstream; numbers; left to right; no base is defined as base zero.

Rates (in E. coli):Transcription: 40 nuc../sec.Similar to rate of translation.

Replication: 1,000nuc./sec/strand

RNA pol creates the‘transcription bubble’ whenIt binds to a DNA. The bubblemoves with it.

Displacing of the product(RNA),reforming the dsDNA

About 17 bp are unwound at any given time.Length of RNA:DNA hybrid within the bubble: up to 12 bp.Length of RNA within the bubble: ~25 b.

Within the transcriptional bubble (in bacteria), RNA Pol :Unwinds and rewinds DNA

Maintains the conditions of the template and coding strands.

Synthesizes RNA.

The transcription reaction can be divided into the Following stages:Template recognition - binding of RNA pol to DNAat a sequence known as promoter forming a “closedcomplex”, unwind the DNA to form an “open complex”,creating the ‘bubble’.

Initiation - synthesis of the first nucleotide bond. RNA polDoes not move while it synthesizes the first ~9 bases. Abortive events may occur, forcing initiation to start again.Initiation phase ends when the enzyme succeeds in extendingthe chain and clears the promoter.

Elongation - enzymes moves along the DNA, extending the RNA, unwinding the DNA exposing new segments of the template and displace the RNA-DNA hybrid to re-formthe original double stranded DNA. RNA emerges as a freesingle strand.

Termination - recognition of the point at which no furtherbases should be added to the chain. The enzyme and theRNA should be released and the DNA re-forms the original duplex state.

Initiation of transcription: a crucial(some time the only) regulatorystep in gene expression.Some key questions:How starting point is recognized?How initiation rate is determined?

The process of transcription: the usualcomplementary base pairing process.

The transcription bubble: transientlyand shortly separation of the DNAto single strands.

Template recognition. Closed complex.

Local unwinding: open complex(template strand is available)

Initiation (up to 9 bases that could bereleased; no move)Promoter clearance

Elongation - Movement of the bubble.(inchworm move or fluent?)

Termination:1. Cease addition of nucleotides. 2. Set complex apart.Just like initiation, termination is sequence-dependent. Defines the terminator.

Stages in which the bubble is created

Promoter: The sequence of DNA needed for RNApolymerase to bind to the template and accomplishthe initiation reaction (synthesis of the first nucleotidebonds).

Terminator: The sequence of DNA required for disrupting the bubble and reforming the DNA duplex(after the last base is added).

an ’ subunits havea channel for the DNA

Yeast RNA Pol II is composed of 12 subunits (holoenzyme). Two subunits form a different sub-complex. Two subunits are not essential for viability.

Following DNA binding and melting, the “clamp” swings back to force a turn. [note, colors of subunits are the same as in the crystal structure]

“wall” protein is enforcinga turn.The length of RNA hybridis limited by the activity ofthe “rudder” protein. TheRNA is forced to leave the DNAWhen it hits the protein rudder.

The bridge protein is found in different conformationsIn different crystal structures.

Probably, breaking and re-making of contactsis mediated by conformational changesof the “bridge” protein:

A nucleotide addition cycle:1. The bridge is in a straight conformation adjacent tothe nucleotide entry site.2. After adding a nucleotide to the RNA the bridge protein is in contacts with the newly added nucleotide, undergoes a conformational change and moves one basepair along the template, obscuring the nucleotide entrysite. 3. The bridge returns to its straight conformation, allowingEntry of next nucleotide of the template - namely,the bridge acts as a ratchet.

The core enzyme of E. coli has a general affinity for DNA (drivenby electrostatic attraction between the basic protein andthe acidic DNA). Yet, it does not distinguish between promoters andother sequences.

Any random sequence bound by core enzyme is describedas a “loose binding site”. No change occurs in the DNAwhich remains duplex.

Such a core enzyme-DNA complex is stable (half life fordissociation is 60 min.).

Properties of the core enzyme

The holoenzyme has a drastically reduced abilityto recognize “loose bindingsites” (half life of <1sec. Kdreduced by a factor of 104).

The holoenzyme binds promoters with Kds 1,000time higher than core enzyme with half lives of hours.However, it manifests aspecific Kd to any specificpromoter.

Sigma confers the ability torecognize specific sites. It isalso involved in “melting”, creating an “open” complex.

Properties of the holoenzyme

Depending of specific promoter the Kd for DNA:RNA polassociation is 106 - 1012 (first level of regulation of rate oftranscription).

Formation of an open complex by melting (that is driven by sigma) allows tight binding that is not reversible.

Initiation rate (frequency of initiation) also differs (dependent on other factors in addition to RNA pol:DNA associatio. Frequencies can range between 1/sec (rRNA genesto 1/30 min. (lacI promoter).

Sigma factor is recycled.It becomes unnecessary whenabortive initiation is concluded.

The holoenzyme binds promoters with Kds 1,000time higher than core enzyme with half lives of hours.This property assists with promoterrecognition, but significantly interfereswith elongation. Therefore, sigma dissociatesfrom the enzyme when elongation starts.

(sigma)

(promoterregion)

Sigma contacts mainly bases of the coding strand and continues to hold these contacts - an important step in melting (forming an “opencomplex and recognition of template strand.

What is responsible for the ability of holoenzyme to bind specifically to promoters?

Sigma has domains that recognize promoter DNA, but as an independent proteinSigma does not bind to DNA. There is major change in conformation of sigmawhen it binds core enzyme. The N-terminal region of free sigma suppresses the

activity of the DNA-binding region - it is an autoinhibitory domain.

How holoenzyme finds a specific promoter (60bp in a 4x106 stretch)?

The forward rate constant for RNA Pol binding to promoters is faster thanrandom diffusion (that limits the constant to 108/M-1Sec-1).

The measured rate constant for association with a 60 bp target is 1014/M-1Sec-1.

If the target is the whole genome the rate constant is around 1014/M-1Sec-1.But how does the polymerase move from random binding sites to promoters?

Perhaps RNA Pol binds DNA and remains contact (no simple diffusion that relies on random binding). Rather, a directDisplacement with other sequence occurs (no sliding).

The “diffusion model: random association with loose sites on DNA,dissociation and re-bind, until occasionally (statistically) interacting with a promoter, and remains associated.

A direct displacementmodel - diffusion is notrequired

Promoter’s function is provided directly byits DNA sequence/structure (it does not need to betranscribed or translated).

It is a cis-acting site.[in genetic terminology, sites that are located on thesame DNA are said to be in cis. Sites that are locatedon two different molecules of DNA are being in trans.]

Conserved - a base most often present at a position.Perhaps the most striking feature of E. coli promoters is the lack of extensive conservationof sequence over the 60 bp associated with RNA Pol.

Promoter elements (in E. coli):Start point (a purine in 90% of the RNAs).-10 sequence-35 sequenceThe distance separating the -35 and the -10 sites.

The -10 sequence:

T80A95T45A60A50T96

Sequence that resides in poistions of -18 to -9 in all known E. coli promoters.

Subscripts denote the percent occurrence of the most frequent foundbase

The -35 sequence:

T82T84G78A65C54A45

The distance separating the -35 and -10 sites is between 16-18 bpin 90% of promoters. In the exceptions it can go down to 15 or upto 20. Sequence itself is not important.

Some promoters have an A-T-rich sequence located farther upstream.It is called UP element and interacts with a subunit of RNA pol. ItIs typically found in promoters that are highly expressed, such as thepromoters of the rRNA genes.

Up elements are associated with a subunit of RNA pol. Found in promoters thatare highly expressed.

In spite of conservation of promoters there is ~1000 fold variationin the rate at which RNA polymerase binds to different promotersin vitro.

Binding rates correlate well with the frequencies of transcription in vivo.

Sequences at prokaryotic terminatorsshow no similarities.

Many terminators require a hairpin toform.

Termination involves recognition ofsignals on the transcript.

Intrinsic terminator - other factorsare not required. Works in vitrotoo.

Hairpins may cause polymerase to slow or even to stop.

Antitermination process may allow RNA Pol to continue(readthrough).

Downstream U-rich destabilizes RNA-DNA hybrid.

Hairpin structure+ U rich sequence

(1100 sequences inE. Coli fit these criteria.

Hairpin + U-rich areNecessary, but not sufficient.

The weakest base-pair is the rU-dA

Rho:A 275 Kd homo-hexamer.RNA binding domain + ATPase domain.Belong to a family of ATP-dependent helicases.

Functions as an ancillary factor for RNA Pol.Most efficient at 10% concentration.Accounts for about 50% of terminations in E. coli.

Rho-dependent termination sequences are rich inCs and poor in Gs. Reside 50-90 bases from termination sites.

Acts processively on a single RNA substrate.

Moves faster than RNA Pol.

Pausing is important for Rho-dependent terminationtoo.

TranslationComponents involved in translation account for 35% of the dry weight of E. coli cells.

A condensation reaction: formation of the peptide bond by removal of water (dehydration) from the -carboxyl group of one amino acid and

the -amino group of another-----------------------------------------------------------------------------------

To make the reaction thermodynamically more favorable, the carboxyl group must be chemically modified or activated so that the hydroxyl group can be more readily eliminated

(Dihydrouridine)

First stage in translation: aminoacyl-tRNA synthetases esterifythe 20 amino acids to their corresponding tRNA.

Each enzyme is specific for one amino acid and one or more tRNAs.

Step 1: An enzyme-bound intermediateAminoacyl-AMP forms when the carboxylgroup of the amino acid reacts with the-phosphoryl group of ATP, creating ananhydride linkage, with displacement ofpyrphosphate.

Step 2: The aminoacyl group istransferred to its corresponding tRNA.The resulting ester linkage has a highly negative standard free energy of hydrolysis.

Valine and isoleucine differ in only a single methylene group

Proofreading by aminoacyl-tRNA synthetases

Two active sites in the Ile-tRNAIle synthetase:

- binding of the amino acid to the enzyme (affinity to Ile is only a little higher than affinity to Val (error in 1/200 entries.

- binding of aminoacyl-AMP product. This site has higher affinity to AMP-Val. A hydrolytic site.

What is accomplished by aminoacylation of tRNA?

1. Activation of the amino acid for peptide bond formation.

2. Attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide.

N-formyl group is added to theamino group of methionine by transformylase.

Transformylase is specific toMet attached to tRNAfMet

Translation initiation in prokaryotes

IF-3 prevents combining of the 30S and 50S subunits

The initiating 5’AUG is guided to its correctposition by the Shine-Delgarno sequence in the 5’UTR of the mRNA (AUG is thebeginning of an ‘open reading frame’).

The initiating 5’AUG is positioned at a sitecalled the P site, the only site in the ribosometo which fMet-tRNAfMet can bind.

The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site.

Step 2 in translation initiation:

GTP-bound IF-2 and the fMet-tRNAfMet join the ribosome, guidedby the anticodon that pairs with the mRNA initiation codon.

Step 3 in translation initiation:

The complex (30S + IF1,IF2-GTP,IF3 + fMet-tRNAfMet) combines withthe 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi which are released from the complex. All 3 initiation factors are also released from the complex.IF-2-GDP is re-loaded with GTP via a GDP/GTP exchange reaction.

Translational elongation

Step 1:Appropriate incoming aminoacyl-tRNAbinds to a complex of GTP bound EF-Tu.

The GTP-EF-Tu-aminacyl-tRNA complexbinds the A site of the 70S complex.

The GTP is hydrolyzed and the EF-Tu-GDPis released.

EF-Tu-GTP complex is regenerated via aGDP/GTP exchange reaction catalyzedby EF-Ts.

The -amino group of the aminoacid in the A site acts as a nucleophile, displacing the tRNAin the P site to form a peptide bond.The tRNAfMet at the P site is nowuncharged.

The peptidyl transferase reaction is probably catalyzed by the 23S rRNA

Translation elongation, Step 2: Formation of the peptide bond:

Move of the ribosome. The ribosomemoves one codon towards the 3’ end ofthe mRNA.Translocation is catalyzed by EF-G-GTP (translocase).

The ribosome is now ready for the next elongation cycle.

Translation elongation, Step 3: Translocation

TerminationCatalyzed by RF1 or RF2. (depending on the particular stop codon).RF1 and RF2 are proposed to mimic thestructure of tRNA.RF-1 recognizes UAG and UAA. RF-2Recognizes UGA and UAA.In eukaryotes, a single RF, eRF, recognizes all 3 termination codons.Releasing factors:1. Hydrolyze the ester linkage of the peptydil-tRNA bond.2. Release the polypeptide and the last tRNA (now uncharged).3. Dissociate the ribosome to 30S and 50S subunits.

EF-Tu EF-G

The carboxy terminal domain of EF-G mimics the structure of tRNA. Altogether EF-G mimics the structure of EF-Tu-tRNA complex and probably binds to the A site and displacing the peptidyl-tRNA.

Translation is energy consuming:

On average, hydrolysis of more than 4 NTPs to NDPs is required for the formation of each peptide bond of a polypeptide.

Bacterial ribosome’s M.W.: ~2.7 million

Components in the ribosome structure:Proteins: blue (in large subunit); Yellow (in small subunit). Bases of rRNA in large subunit: white. Backbone of rRNA in large subunit: green. rRNA in small subunit: white. tRNAs: purpule, mauve, gray. mRNA contacting tRNAs: red.

In the 50S subunit, the 5S and 23S rRNAs form the structural core.The proteins are secondary elements in the complex, decorating thesurface.

No protein is detected within 18A of the active site for peptidebond formation.

50S subunit of a bacterialribosome.Red - a puromycine moleculeat the active peptidyl transferase site. Noteno proteins in the vicinity.

Steady state level of a protein (expression level) is determinedby a combination of regulation of:

Transcription initiation

mRNA degradation (mRNA stability)

mRNA processing

Transport to cytoplasm

Translational control

Folding and protein processing

Protein degradation (protein stability)

Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: engelber@cc.huji.ac.il

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