Expression of Genome

Chapter 13 Mechanism of Transcription Chapter 14 RNA Splicing

Chapter 15 Translation Chapter 16 The Genetic Code mRNA, tRNA, and attachement of amino acids to tRNA The ribosome and genetic code Translation initiation, elongation, and termination Regulation of translation Translation-Dependent regulation of mRNA and protein

Introduction1. Proteinsend products of most information pathways.

Requires thousands of different proteins at any moment.

2. Eukaryotic protein synth involves >70 different ribosomal proteins; >20 enzymes to activate AA precursors; a dozen or so auxiliary enzymes and other protein factors for initiation, elongation, termination. ~100 additional enzymes for final processing; and >40 kinds of tRNA and rRNAs. Overall, ~300 macromolecules cooperate to synthesize polypeptides.

3. In typical bacterial cells: Total of 15,000 ribosomes, 100,000molecules of related protein factors/enzymes, and 200,000 tRNA(15% total RNA) molecules, account for more than 50% of the cell’s dry weight.

4. In rapidly growing cells, protein synthesis account for up to 80% of energy used. Every cell contains several to thousands of copies of many different proteins and RNAs.

5. Despite great complexity, proteins synthesized at exceedingly high rates. In E. Coli, at 37 °C about 20 aa/sec, as 3 bases/aa, comparable to transcription (50-100 base/sec). In eukaryotic, at 2-4 aa/sec, as separation of transcription and translation.

6. Synthesis of proteins is tightly regulated, just enough copies are made to match current metabolic needs.

7. Also, to maintain the appropriate concentration of proteins, the targeting and degradative must keep pace with synthesis.

8. The study of protein synthesis offers important reward: a look at a world of RNA catalysts that may exist before the dawn of life. Proteins are synthesized by a gigantic RNA enzyme!

How protein were made?it’s unlikely direct interactions between mRNA and the amino acids be responsible for specific and accurate ordering of amino acids in a polypeptide.

RNA in Information Transfer

Information from nucleus-DNA to cytoplasm-ribosomes in eukarycells. However, information transfer not clear until mRNA shown to exist. Far from straightforward.

• Until ~1960, thought rRNAs as templates for protein synthesis

• François Jacob and Jacques Monod questioned, because rRNAs are homogeneous in size (5/16/23S in bacteria, whereas mol wts of proteins vary over at least two orders of magnitude.

• By analyzing E. coli mutants altered in control lactose metabolism (WT, constitutive or non-inducible), Jacob and Monod predicted existence of mRNA, another RNA species from DNA template as protein template.

• ie, rapid changes β-galactosidase-forming capacity, suggested mRNA metabolically unstable—(ie, control at translational).

• Because rRNAs/tRNAs are quite stable, unlikely labile intermediates

• It constitutes only a small proportion of total RNA (1-3% in bacteria), mRNA presence masked by much more abundant rRNAs/tRNAs.

The Genetic Code

Three major advances

Ribosomes and endoplasmic reticulum.

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1. Early 1950s, Paul Zamecnik, radio-AA into rats, at different time intervals, examined sub-cellular fractions for radioactive protein.

After hours/days: all subcellular fractions w labeled proteins.

After only min.: labeled protein only in a fraction containing small ribonucleoprotein particles

These particles, visible by EM, site protein synthesis from AAs, later named ribosomes.

Three major advances2. Second, Mahlon Hoagland/Zamecnik, AAs “activated”

when incubated w/ ATP and cytosolic fraction of liver.• AA attached to a heat-stable soluble RNA, later known as tRNA

(representing 15% cellular RNA), form aminoacyl-tRNAs.

• Enzymes catalyze the process are aminoacyl-tRNA synthetases.

3. Third advance, Francis Crick’s genetic information in 4-letter language (A-C-T-G) translated into 20-letterlanguage of proteins (20-AAs).• A small nucleic acid (--- RNA) as adaptor, part of adaptor

binding a specific AA, another part recognizing nt seq code that AA in mRNA.

• This idea verified, tRNA adaptor “translates” the nucleotide sequence of mRNA into amino acid sequence.

• Overall referred to as translation.

The RNA Tie Club (necktie with an embroidered RNA helix and 3-lett aa-tiepin), and Crick’s adaptor hypothesis

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Amino acyl tRNAsynthetases

Links 20-AAs/4-nts

thought to fix AAs directly onto DNA sequence,

Crick argued the incompatibility on physical-chemical basis.

• 1960s, apparent at least three nt residues of DNA necessary to encode each AA. The 4-letters (A, T, G, and C) in groups = 2, yield 42 = 16 combinations, <20 AAs. In Groups = 3, yield 43 = 64 different combinations.

Key properties of genetic code1. Translation occurs that these nt triplets are read in a

successive, nonoverlapping fashion.

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The Genetic Code

Overlapping versus nonoverlapping genetic codes

2. General nature of genetic code from analyzing on the effects of deletion and insertion mutations.

Key Properties of the Genetic Code

3. A specific first codon in sequence establishes reading frame, in which new codon begins every 3-nt residues.

4. Each reading frame gives a different sequence of codons, only one is likely to encode a given protein.

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How the Code Was

Cracked?

Breakthrough:• 1961, Marshall Nirenberg and Heinrich Matthaei

Incubated poly(U) +E . coli extract, GTP, ATP, and mixture of 20 AAs in 20 different tubes, each tube a different radio-AA

poly(U)-mRNA synth. polypeptide w/ AA encoded by UUU.

A radioactive polypeptide indeed formed in only one of the 20 tubes, the one containing radioactive phenylalanine.

• Similarly, revealed poly(C), CCC Proline, poly(A), AAA Lysine.

• Poly(G) did not, as it forms tetraplexes bound by ribosomes.

• next, using mixed nucleotides, eg, 5x ADP and 1x part CDP: AAA >> AAC/ACA/CAA > ACC/CCA/CAC >> CCC

Diligence and Chance produce a breakthrough —

the experiments should not have worked: absence of initiation codons.

Luckily, high [Mg2+] used, circumvent the need of IFs and specific initiator fMet-tRNA.

1964, another breakthroughs1. E. coli ribosomes bind specific aminoacyl-tRNA in the presence of

the corresponding polynucleotide messenger.

ie, ribosomes incub. poly(U) bind PhetRNA-Phe) only but not other.

Nirenberg and Philip Leder

• Later, found a trinucleotide UUU sufficient to bind

• w/ this, trinucleotide tech, 50/64codons identified. But not all, some bind weakly or not specifically.

2. By H. Gobind Khoranamethods to synthesize polyribonucleotides w/ defined, repeating 2-4 nt bases

The copolymer (AC)n, eg, ACACACACAC… alternating ACA/CAC codons get equal Thr/His.

Known that His = codon 1A2C, CAC must for His, and ACA for Thr.

Genetic codon cracked

• All codons by 1966, one of the most important discoveries of the 20th century.

• A special initiation beginning codon AUG in all cells, in addition

to Met residues.

• Termination (UAA, UAG, and UGA), stop or nonsense codons, not code any known AAs.

• In random nt sequence, average 1/20 termination codons in a reading frame.

• Thus, a reading frame w/o a stop ≥ 50 codons, as an open reading frame (ORF). Long ORF usually protein encoding genes.

• A striking feature of the genetic code is an AA may be specified by ≥ 1 codon, as degenerate of codon.

Degeneracy genetic code

The 1st base of codon in mRNA (5'→3') pairs w the 3rd base of anticodon in tRNA. Wobble allows some tRNA to recognize >1 codon: 3 diff mRNA codons-tRNA

anticodon has inosinate (I). The 5’-1st base-tRNA anticodon determine the number of recogn codons. minimum of 32 tRNAs required to all 61 codons (31-for AAs and 1-for initiation). Specificity by the first-2 bases

of mRNA codon

The wobble hypothesis:Exam codon-anticodon pairings conclude the 3rd base of most codons pairs loosely with its anticodon --wobble hypothesis.

Natural Variations in the Genetic Code• variations in code in some organisms, mostly in mitochondria,

(which encodes 1020 proteins)

• A few proteins in all cells (e.g. formate deHase in bacteria and GPx in mammals, ~25 in mammals) require selenium, generally as selenocysteine.

• A special serine-tRNA, recognizes UGA codon. It is serine-charged andenzymatically converted to selenocyste.

• In effect, cells has 21 common AAs, and UGA can be a codon for terminationand (sometimes) selenocysteine.

Mutations of the Genetic Code• Alteration changes a codon specific for one AA to for another AA =

missense mutation.

• A more drastic alteration causing a chain-termination codon, known as a nonsense or stop mutation.

• The third kind of point mutation is a frameshift mutation.

• Sometimes, the harmful mutations can be reversed by a second genetic change.

1)The simple reverse (back) mutations (the same site).

2)The suppressor mutations (additional mutation-B suppressed the original mutation-A): If in the same gene (intragenicsuppression) and those in different gene (intergenicsuppression).

• Genes that cause suppression of mutations in other genes are called suppressor genes

2. Intergenic suppression (between mRNA/tRNA)

• The Nonsense suppression by a minor tyr-tRNA (anticodonGUACUA), to suppress the nonsense mutation generated UAG.

• Suppression of UAG is efficient, but UAA suppression is poor (only 1-5%). Because in E coli, UAG not frequently used stop codon (most used is UAA).

• Also, E coli producing UAA-suppressing tRNAs grow poorly.

WT tyr-tRNA

Nonsense suppressive mut tyr-tRNA

The Ribosome Is a Complex Supramolecular Machine

1. Each E. coli contains ≥15,000 ribosomes, making up ~25% dry weight of cell2. Bacterial ribosomes 65% rRNA and 35% protein; diameter ~18 nm, 3. First found in 1941 by A. Claude. By 1956, H. Schachman, yeast, 80S 60S/40S

(reversibly dissociate, diameter 23 nm), later A. Tissieres and J. Watson simliarly in E coli, 70S 50S/30S.

4. 1968, Masayasu Nomura: showed rRNA and protein spontaneously reassemble form 30S or 50S subunits.

Ribosome subunits and sedimentation coefficient

The ribosome cycle

Rapid Translation by Polysome: a ribosome synthesize only one polypeptide at a time, but each mRNA can be translated simultaneously by multiple ribosomes (10-100).Explains relatively limited abundance of mRNA in cell (1%–5% total RNA)

The Large/Small Subunits Undergo Association/Dissociation during Each Cycle of Translation

Ribosome three tRNA-binding sites (E, P and A) each span two sub-units

Polypeptide exit tunnel in 50S subunit, rRNA white and proteins yellow,

The red and gold of rRNA adjacent to A-site tRNA are peptidyl transferase center.

peptidyltransferase center

The peptidyl transferase

Reaction, peptide formation

by hydrolysis of high-energy

acyl bond in AA-tRNA

rRNAs, Structural/Catalytic Determinants of the Ribosome

E PA

The close proximity of 3’- ends of A-site and P-site tRNAs

EP A

The peptidyl transferase

Center (no protein w/i 18 Å )

proteins as secondary elements, most globular, some snakelike protein extensions protrude into rRNA core, stabilizing its structure

EP

A

The decoding center

Characteristic Structural

Features of Transfer RNAs

Characteristic Structural Features of tRNAs

1. tRNAs, adaptors in translating nucleic acids into proteins.

2. relatively small, 73 - 93 bases, 24-31 kd, ssRNA folded a precise 3D.

3. at least 1-tRNA/ea-AA; at least 32 tRNAs for all normal codons,

4. Mitochondria and chloroplasts, smaller tRNAs. Vertebrate mtDNAsencode 13 proteins, 2 rRNAs, and 22 tRNAs. Unusual wobble allows the 22 tRNAs to decode all 64 codons.

• Either U pairs with all 4 possible bases in 3“two out of three” mechanism—no base pairing at 3rd position.

5. >8 modified bases, methylated bases, , pseudouridine; T, ribothymidine; D, 5,6-dihydrouridine

6. Most tRNAs 5’-pG /CCA-3’ (CCA-adding enzyme).

7. All tRNAs-cloverleaf structure, five arms;

the 5th arm-variable loop (extra arm, 3-21 bases).

8. G=U base pairs, determinant for Ala-tRNA synthetase

non-Watson/Crick pairing.

Yeast tRNAAla

The secondary structure of tRNA.

• The acceptor stem, CCA- 3’ site for amino acid, by pairing between 5’ and 3’ ends of tRNA molecule.

• The U loop, of unusual base U in the loop. often found within sequence 5’-TUCG-3’.

• The D loop, presence of dihydrouridines.

• The anticodon loop, contains anticodon, for pairing codon. The anticodon always bracketed on 3’ by a A/G and on 5’ by U.

yeast tRNA-Phe

partially stacked

Protein Biosynthesis in Five Stages

•Stage 1: Activation of Amino Acids• Enzymatic synthesis of aminoacyl tRNA molecules

•Stage 2: Initiation• Binding of mRNA and N-formylmethionine to ribosome

•Stage 3: Elongation– Binding of aminoacyl tRNAs to ribosome– Formation of peptide bonds

•Stage 4: Termination and Ribosome Recycling– Termination codon in mRNA reaches ribosome

•Stage 5: Folding and Posttranslational Processing

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Stage 1: AA-tRNASynthetases Attach the

Correct AAs to Their tRNAs

AA-tRNA in Ester linkage

1. carboxyl- of AA must be activated,

2. In cytosol, not ribosome.Each AA covalently attached to a specific tRNA, (need ATP, Mg2+-dependent AA-tRNAsynthetases.

3. When aminoacylated, the tRNAs are “charged.”

• The 20 AA-tRNA synthetases divided into two classes on 1/3structure and in reaction mechanism

• These two classes, same in almost all organisms. no evidence for a common ancestor for them.

• An exception, some bacteria lack Gln-tRNA, but use Glu-tRNAand then convert Glu Gln.

Aminoacyl-tRNA synthetases

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Bound ATP (red) pinpoints the active site near the end of the aminoacyl arm.

(a)monomeric Class I synthetase: Gln-tRNA synthetase from E. coli.

(b)dimeric Class II synthetase : Asp-tRNA synthetase from yeast

ATP

Class I enzymes, initially to 2’-OH, then 3’-OH by transesterificationClass II enzymes, transferred directly to 3’-OH of terminal adenylate.

Rapidly equilibrium

• The specificity determinants are clustered at two distant sites : the acceptor stem and the anticodon loop.

• Some positions (blue dots) are the same in all tRNAs and cannot be used to discriminate tRNAs.

tRNA Synthetases Recognize Unique Structural Features of Cognate tRNAs

• known recognition points for one (orange) or more (green)aminoacyl-tRNA synthetases.

• Only a few nts in tRNA may confer binding specificity, eg, G=U in Ala-tRNA synthetase

• Structural features other than sequence are important.

Ala-tRNAsynthetases is a single G=U

base pair

acceptor stem

Stage 2: Initiation of Translation

Protein Synthesis begins at the N-terminal end

1. Protein synthesis begins at N-terminal end, by stepwise addition AA to C-terminal of the growing polypeptide, (Howard Dintzis, 1961)

Reticulocytes incubated with radioactive leucine. Samples of completed chains were isolated at various times afterward, and the distribution of radioactivity was determined (brown).

Radioactivity terminal

AUG initiation codon• AUG initiation codon specifies an N-terminal methionine

• all organisms have two tRNAs for methionine, In bacteria, tRNAMet and tRNAfMet.– One for initiating-AUG codon, the other for Met internal AA.

• N-formyl-methionine in initiating-AUG codon is (fMet), by 2-steps:

• 1st, met-charged same Met-tRNA synthetase for tRNAfMet/tRNAMet

• Next, a transformylase (specific for Met on tRNAfMet) transfers a formylfrom N10-formyl-THF to amino of Met on tRNAfMet :

• In eukaryotes, begin w Met (not fMet), but also a specialized initiating tRNA.• Mitochondria/chloroplast still begins w/ N-formylmethionine, removed after

synthesis.

Three Initiation Factors in Assembly of Initiation Complex Containing mRNA and ini-tRNA

1. The mRNA binds to smaller subunit and the initiating aminoacyl-tRNA. Prokaryotic mRNAs Initially recruited to small subunitby base pairing 16S-rRNA.

2. The large subunit then binds an initiation complex.

3. The initiating AA-tRNA base pairs w/ the mRNA codon AUG, signals beginning .

4. This process, requires GTP, promoted by initiation factors (IFs).

• The initiating AUG guided to correct position by S-D seq in mRNA.

• The S-D sequence bp w/ a pyrimidine-rich sequence near the 3’ end of 16S rRNA of the 30S subunit

• S-D sequence: is an initiation signal of 4-9 purine residues, 8-13 bpto the 5’ side of the initiation codon.

Shine-Dalgarno sequence

The Steps of Initiation1. The initiation requires: the 30S ribosomal subunit, mRNA for the polypeptide the initiating fMet-tRNAfMet, Three initiation factors (IF-1, IF-2, IF-3), GTP, the 50S ribosomal subunit, Mg2+.

2. 30S ribosomal subunit binds IF-1/ IF-3. IF-3prevents 30S and 50S subunits combining prematurely.

3. Both subunits contribute to A and P sites, But E site is largely in the 50S subunit, for “uncharged” tRNAs leave.

4. Factor IF-1 binds A site, prevents tRNA binding at this site during initiation.

5. IF2, a GTPase, facilitates association of fMet-tRNAi

fMet w/ small subunit and prevents other charged tRNAs from associating.

6. The initiating AUG at P site, the only site fMet-tRNAfMet can bind, as fMet-tRNAi

fMet

is the only AA-tRNA binds directly to P site;

7. All other AA-tRNAs (including the interior Met-tRNAMet) bind first to A site, then to P/E

8. The last step, association of the large subunit 70S initiation complex. IF3 release by conformational change.

9. w/o IF3, IF2 acts as docking site of the large subunit, stimulates GTPGDP, IF2-GDP loss affinity, departs with IF1. Now, mRNA with fMet-tRNAi

Met in P site and an empty A site.

10. The initiation complex now ready for elongation.

Translation initiation

Translation in Prokaryotic and Eukaryotic

messages

Structure of mRNA a) Polycistronic prokaryotic message with 3-ORFs and Ribosome binding site. (b) A monocistronic eukaryotic message, 5’-Cap.

Eukaryotic Ribosomes Are Recruited to mRNA by 5' Cap

1. eukaryotes, small subunit associated ini-tRNA when recruited to capped 5'-mRNA

2. "scans" along mRNA in 5’3’,to first AUG fit Kozak sequence.

3. Eukaryotes require more auxiliary proteins. In 4 steps:

1) binding of ini-tRNA to small subunit precedes the mRNA.

2) separate set of auxiliary factors mediates mRNA recognition.

3) ribosome bound ini-tRNA scan for the first AUG in mRNA .

4) the large subunit is recruitedafter ini-RNA pairs to start codon

43S PIC

48S PIC

eIF4B activates RNA helicase activity of eIF4A to remove mRNA 2nd structure

eIF4E binds Cap

Translation Initiation Factors Hold Eukaryotic

mRNAs in Circles

• Initiation factors closely associated with 3' end of mRNA through its poly-A tail, between eIF4G (5’-mRNA/CAP-bound) and (PABP).

• Both elF4G and the PABP bound to mRNA through multiple rounds of translation.

• Provides explanation for the observation of poly-A tail contributes to efficient translation of mRNA.

A model for the circularization of eukaryotic mRNA. Circularization is proposed mediated by an interaction between eIF4G and the poly-A-binding protein (PABP)

The 48S initiation complex. mRNA as a black line with yellow bar for coding region, and the 5'-cap is a black circle.

The Kozak sequence

The Kozak sequence must not be confused with the ribosomal binding site (RBS), that being either the 5' cap of an mRNA or an Internal Ribosome Entry Site (IRES).

Start Codon by Scanning Downstream from 5‘-mRNA

Identification of the initiation AUG by 48S PIC and large sub joining in eukaryotic initiation of translation

•48S PIC

•Scan for AUG, drive by eIF4A/B-asso RNA helicase, ATP, 5’3’

•Once paired, conformational change eIF5/eIF2, GTP GDP, release factors

•allows binding of GTP-eIF5B, binds ini-tRNAstimulates asso60S subunit. 80S initiation

complex

Joining of 60S subunit

Driven by ATP hydrolysis, eukaryotic initiation complex scan the mRNA until the first AUG is reached.

uORFs and IRESs : Exceptions that Prove the Rule

The first ORF not always the real one. uORF usually encode polypeptides <10 aa. uORF act to regulate the extent of translation, generally reduce but not eliminate translation of the long downstream ORF.

<50% of 40S subunit retained,

scan for 2nd AUG, restart

uORF

IRESs (internal ribo re-entry sites) bypass normal requirement of translation initiation as eukaryotic RBS

Different IRES seq by different mech.

The most extreme case: Cricket paralysis virus mRNA mimic tRNA-bound-mRNA, 40S binds directly to P site, bypass the need of all IFs and ini-tRNA

Cricket paralysis virus mRNAmimiv cap-binding eIF4E

Stage 3: Translation Elongation 1. Peptide bond formed in elongation.

2. E coli, requires The initiation complexaminoacyl-tRNAs, 3 elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), GTP.

3. Three steps to add each aa• First, correct AA-tRNA loaded to A site dictated by A-site codon.

• Second, peptide bond formed between AA-tRNA in A and peptide chain attached to peplidyl-tRNA in P site.

• Third, resulting peptidyl-tRNA associated codon in A site translocated to P site, poised for another cycle of elongation.

4. The elongation conserved between prokaryotes and eukaryotes. 3- eukaryotic elongation factors [eEF1a (EF-Tu), eEF1bc (EF-Ts), and eEF2(EF-G)] functions analogously.

Step1: EF-Tu escorts AA-tRNA to A site .

• AA-tRNAs not bind to ribo. on their own. Instead, they are "escorted" to ribosome by EF-Tu.

• Once tRNA is aminocylated. EF-Tu-GTP(EF-Tu-GDP, or EF-Tu alone, little affinity for AA~tRNAs) binds to 3’-end tRNAs, masking the coupled-AA.

• Charged tRNAs bound to EF-Tu-GTP as they first interact with A site. When correct codon-anticodon interaction occurs, EF-Tu interacts with factor-binding center, hydrolyzes GTP to GDP, and released from tRNA and ribosome.

The Ribosome Is a Ribozyme• Proof that peplidyl transferase entirely composed of RNA from

high-resolution, 3-D structure of ribosome, reveals that no AA located closer than 18 Å from active site.

• Because catalysis requires distances in the 1-3 Å range. it is clear that the peptidyl transferase center (in 23S or 28S large sub) is a ribozyme. Thus, ribosome is a huge ribozyme.

• Mutation or deletion of L27 protein, the only protein that is close enough to peptidyl transferase center, still retained majority 30-50% of activity. Thus, the role of L27 is to correctly position the active site of RNA component.

• Elimination of 2’-OH of A2451 of 23S rRNA reduce by 10x act.

• Recent study: P site tRNA, 2’-OH of the CCA tail is critical, as its’ mutation cause 106-fold reduction in catalysis rates. Substrate-assisted catalysis. before ribosome, tRNA catalyze protein synthesis.

no AA located closer than 18 Å from active site

P site tRNA,

CCA-2’-OH

Step II: Peptide bond formation• The a-NH2 of the A site AA as a nucleophile,

displacing the P site tRNA to form peptide bond.

• The peptidyl transferase catalyzing reaction is the P-site tRNA ribozyme.

Step 3: Translocation

Ribosome moves one codon toward 3’ end of mRNA. As EF-G-GTP GDP, (note the conformational change ).

Shifts anticodon of the dipeptidyl-tRNA, which attached to 2nd codon, from A to P.

Shifts deacylated tRNA from P to E, and then is released.

The 3rd codon now in the A site and open for the incoming (third) aminoacyl-tRNA.

Counterclock wise rotation of small/relative to large subClockwise rotation back

Molecular Mimicry of EF-G Translocase

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Ribosome movement using energy provided by hydrolysis of GTP bound to EF-G (translocase).

EF-G-GDP

C-terminal part of EF-G mimics the anticodon loop of tRNA in both shape and charge distributionEF-G bind the A site and displace the peptidyl-tRNA

EF-Tu–tRNA

• For EF-G-GDP, GDP has a lower affinity for EF-G than does GTP and is rapidly released after hydrolysis. The unbound EF-G rapidly binds a new GTP molecule.

• However, for EF-Tu-GDP, the factor EF-Ts is required, which acts as a GTP exchange factor.

EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to a new round of elongation

A Cycle of Peptide Bond Formation Consumes Two GTP and One ATP

An ATP consumed by AA-tRNA synthetase in charging AA to tRNA.

A GTP consumed in delivery charged tRNA to A site by EF-Tuand ensuring correct codon-anticodon.

Another GTP consumed in EF-G-mediated translocation.

Only ATP is energetically connected to peptide bond formation. The energy of two GTP is to ensure the accuracy and order of events during translation.

Proofreading on ribosome only for proper codon-anticodonpairing. The identity of the amino acid on AA-tRNA not checkedon the ribosome (it is checked in aa-tRNA synthetase step).

Thus, wrong amino acids can be inserted for experiments.

The Ribosome Uses Multiple Mechanisms to Select Against Incorrect Aminoacyl-tRNAs

1st mechanism for fidelity of codonrecognition:

• Additional H-bonds formed between two A residues of 16S rRNA and minor groove of anticodon-codonpair only when correctly bp-ed.

• Net result is correctly paired tRNAsexhibit a much lower rate of dissociation than do incorrectly paired tRNAs.

• 2nd mechanism helps to ensute correct codon-anticodon pairing involves the GTPase activity of EF-Tu.

• Correct pairing allows EF-Tu-bound to AA-tRNA to interact with factor binding center inducing GTP hydrolysis and EF-Turelease.

• A 3rd mechanism ensures pairing accuracy is proofreading after EF-Tu is released.

• the 3’-end of AA-tRNA moves almost 70A˚, rotation places a Strain, mispaired tRNAs dissociate from the ribosome.

• Only correctly bp-ed AA-tRNAs remain associated with ribosome as they rotate into the correct position,

• The rotation is referred to as tRNA accommodation.

tRNA accommodation

Stage 4: Termination1. Termination, signaled by stop

codons.

2. Prokaryotes, two class I RFs I: RFland RF2. RF-1 recognizes the UAG and UAA, and RF-2 recognizes UGA and UAA. In eukaryotes, a single eRF, recognizes all three stop codons.

3. Class I factors stimulate polypeptide release through a conserved GGQ (gly-gly-gln) motif localized the peptidyl transferase center. The SPF (Ser–Pro–Phe) motif to bind anti-codon.

4. Prokaryotes and eukaryotes have only one class II factor, RF3 and eRF3. Like IF2, EF-G, EF-Tu, RF3 is regulated by GTP.

GDP/GTP Exchange and GTP Hydrolysis Control the Function of the RF3

• RF3 is a GTP-binding protein, but, unlike other GTP-binding proteins in translation, RF3 has higher affinity for GDP than GTP. free RF3 predominantly GDP-bound.

• After polypeptide release, a change in ribosome and RF1/2 conformation stimulates RF3 to exchange bound GDP for GTP (GTP-RF3 leads to high-affinity interaction w/ ribosome that displaces class I factor).

• This interaction stimulates the hydrolysis of GTP. Without a bound class I factor, RF3-GDP has a low affinity for the ribosome and released.

3D structures of RF1 bound to the ribosome.(a) RF1 binding to the A site of ribosome.(b) the peptide anticodon is located very near stop codon. (c) RF1 bound to ribosome the GGQ motif located close to 3' end-CCA of P-site

tRNA and the peptidyl transferase center.

GGQ

Comparison of the structures of RF1 to a tRNA. The tRNA is shown

in dark red and RF1 is in gray.

RF1 have domains mimic the structure of tRNA

FIGURE 14-38

Ribosome recycling factor (RRF), EF-G, and IF3 combine to stimulate the release of tRNA and mRNA from a terminated ribosome.

RRF is a mimic of tRNA. it resembles a tRNA in i'ts 3D structure.

Ribosome recycling

Eukaryotic translation termination and

ribosome recycling

• eRF3-GTP escort eRF1 to ribosome for termination

• GGQ motif into peptidyltransferase center leading to polypeptide release

• Rli1: eRF1 in conjunction with an ATPase

Post-translational modifications of proteins1. Amino-Terminal and Carboxyl-Terminal Modifications the N-formyl group, N-Met residue, and often additional N-

terminal (or C-terminal) residues may be removed. In about 50% of eukaryotic proteins, the amino group in N-terminal

is N-acetylated after translation. C-terminal may also modified.2. Loss of Signal Sequences3. Attachment of Carbohydrate Side Chains

• Glycoproteins and proteoglycans4. Addition of Isoprenyl Groups

• ras protein and other proto-oncogenes, and G proteins. The isoprenyl group helps to anchor the protein in a membrane

5. Addition of Prosthetic Groups • Heme, biotin, lipoamide, FAD+, iron-sulfur clusters, etc.

6. Proteolytic Processing Many proteins are initially synthesized• Such as Proinsulin and almost all proteases.

7. Formation of Disulfide Cross-Links

8. Modification of Individual Amino Acids

Phosphorylation: Ser, Thr, and Tyr residues of some proteins; the phosphate groups add negative charges to these polypeptides. • Mostly for signal transduction,

• but also for nutirent : the milk protein casein has many phosphoserines that bind Ca2+. Calcium, phosphate, and amino acids are valuable to suckling young, so efficiently provides three essential nutrients.

Carboxylation : extra carboxyl to Glu residues in mostly , the blood-clotting proteins (require vit K, koagulation, vit K cycle).

Methylation: mono- , di-, and tri-methyl lysine or arginine residues in muscle proteins, histones, cytochrome c, calmodulin, etc. • In other proteins, the carboxyl groups of some Glu residues undergo

methylation, removing their negative charge.

A thioether is formed between the isoprenyl group and a Cys-SH of protein

Regulation of bacterial translation initiation by

inhibiting 30S subunit binding

Intramolecular bp can interfere with mRNA binding to16S rRNA. Mostly, by translation of other genes in same operon. If first ORF is translated, the interfering is disrupted, allowing the other RBS free to bind ribosome and be translated.

Protein encoded by the

mRNA binds to its own RBS

Regulation of Prokaryotic Translation:

Ribosomal Proteins Are Translational Repressors

of Their Own Synthesis

• In addition to transcriptional control, the most important control of ribosomal protein synthesis is at the translational level.

• By autorepression, in each ribosomal protein operon, one (or a complex of two) of the encoded ribosomal proteins binds that operon’s mRNA, interfere with binding of small subunit to RBS.

Protein as translational repressor of

the other proteins is in red.

Promoter in purple

Regulation of ribosomal protein expression

With free rRNA,

Translation OK

no free rRNA,

translation of ribosome

protein blocked

Ribosomal protein S8 binds 16S rRNA and its own mRNA

(a) region of 16S rRNA bound by the S8 protein.

(b)translation initiation site of ribosomal protein S8 that is

bound when no available 16S rRNA to bind.

Box region:

16S rRNA protected

by the S8 protein

S8 AUG

Shared sequences are

shaded in dark green

Upon amino acid starvation, viral infection, and elevated temperature, stresses, two mechanisms of translation inhibition:

1. mediated by phosphorylation of eIF2, leading to reduced levels of eIF2–GTP, thus limit initiation of translation.

2. targets the 5’-cap-binding protein: eIF4E, by phosphorylation of 4EBPs (eIF4E-binding proteins), by a key cellular protein kinasemTOR.

3. Growth factors, hormones, and other factors that stimulate cell division activate mTOR kinase and therefore increase the overall translational capacity of the cell.

Global Regulators of Eukaryotic Translation

Target Key Factors Required for mRNA Recognition and

Initiator tRNA Ribosome Binding

Global Regulators of Eukaryotic Translation

mTOR phosphorylates 4E-BPs

4E-BPs compete with eIF4G for

association with the cap binding protein

eIF4E. This prevents the eIF4A-mediated

unwinding of the mRNA 5’-end and

eIF4G-dependent recruitment of the 43S

preinitiation complex.

Regulation of Ferritin translation by iron

• The 5’-UTR of ferritin genes includes a stem–loop structure: the iron regulatory element (IRE). The iron regulatory protein (IRP) binds tightly to this site when not bound to Fe2+. By stabilizing the stem–loop structure of the IRE, IRP prevents eIF4A from removing this structure from the end of ferritin mRNA. Under these conditions, association of 43S-PIC with mRNA cannot occur and ferritin genes not translated.

• When iron levels elevated and Ferritin protein is needed, IRP binds to Fe2+, which inhibits its ability to bind IRE, therefore, allows translation of Ferritin protein.

mRNAs with premature stop codons are targeted for degradation

For mutant or damaged mRNA, translation is used to detect defective mRNAs and eliminate them and their protein products.

1. In cases when translating of an mRNA lacking the stop codon: In prokaryotes, such stalled or trapped ribosomes are rescued by a chimeric RNA molecule that is part tRNA and part mRNA, appropriately, a tmRNA. The SsrA RNA rescues ribosomes that translate broken mRNAs.

2. In Eukaryotic cells degrade mRNAs that are incomplete or have premature stop codons by several mechanisms.

nonsense-mediated mRNA decay.

nonstop-mediated decay, read into polyA, (poly-lysine residues)

no-go decay: the 3rd mRNA surveillance mechanism related to nonstop-mediated decay.

In prokaryotes, the tmRNA ssrA rescues ribosomes stalled on prematurely terminated mRNAs. The SsrA RNA mimics part of mRNA/part of tRNA (hence tmRNA), it only bind ribosome stalled at the 3‘-end of an mRNA. Once bound, SsrA RNA substitutes part its sequence to act as “mRNA."

How if the ribosome is stalled with no stop codon?

SsrA RNA then

acts as mRNA first acts

as tRNA

1. Nonsense-mediated mRNA decay: In premature stop codon, ribosome released before displacement of all of

the exon–junction complexes. The still bound exon–junction complexes

and eRF3 bound prematurely terminating ribosome recruit and/or activate

multiple enzymes that cleave mRNA.

Translation a normal mRNA displaces

all of the exon–junction complexes

Undisplaced complexes, recruit the Upf1,

Upf2, Upf3 (up-frameshift factors) to the ribosome. Once bound, these

proteins activate enzymes degrading the mRNA.

2. Nonstop-mediated decay

• In the absence of stop codon, mRNA poly-A tail is translated, add poly-lysine.

• Upon reaching 3’-end of template, stalled ribosome recognized by complex Dom34/Hbs1. After delivering Dom34 to ribosome, Hbs1 hydrolyzes GTP and is released.

• In combination with Rli1 ATPase (Rli1: eRF1 in conjunction with an ATPase), Dom34 acts to disassemble the ribosome and recruit an endonuclease that cuts mRNA.

• The resulting mRNA fragments degraded by 5’3’ and 3’5’ exonucleases. The protein with polylysine end is also subject to proteolysis.

3. No-go-mediated decay

• As with nonstop-mediated decay, no-go-mediated decay is initiated when the ribosome stalls.

• In this case, the stall is induced by

an RNA secondary structure or

a stretch of codons demanding charged tRNAs that are present in low abundance (referred to as rare codons).

• The stalled ribosome is recognized by Dom34/Hbs1,

• the ribosome is released and the mRNA degraded in a similar manner to non-stop-mediated decay.

Protein Synthesis Inhibitors

1. Protein synthesis, critically for cells, is the primary target of many naturally occurring antibiotics and toxins.

2. Natural selection in evolution: the minor differences between bacterial and eukaryotic protein synthesis are sufficient that most of the compounds discussed here are relatively harmless to eukaryotic cells (but harmful to mitochodria and chloroplast).

3. Antibiotics and toxin have become valuable tools in the study of protein biosynthesis.

Antibiotics Arrest Cell Division by Blocking Specific in Translation

Antibiotics Arrest Cell Division by Blocking

Specific in Translation

Tetracyclines inhibit protein synthesis in bacteria by blocking the Asite on the ribosome, preventing the binding of aminoacyl-tRNAs.

Chloramphenicol inhibits protein synthesis by bacterial (and mitochondrialand chloroplast) ribosomes by blocking peptidyl transfer; it does not affect cytosolic protein synthesis in eukaryotes.

Cycloheximide blocks the peptidyl transferase of 80S eukaryoticribosomes but not that of 70S bacterial (and mitochondrial and chloroplast) ribosomes.

Streptomycin, a basic trisaccharide, causes misreading of the genetic code (in bacteria) at relatively low concentrations and inhibits initiation at higher concentrations.

Inhibitors of eukaryotic protein synthesis:

1. Cycloheximide

2. Diphtheria toxin (Mr 58,330) catalyzes the ADP-ribosylation of a diphthamide (a modified histidine) residue of eukaryotic elongation factor eEF2, thereby inactivating it.

3. Ricin (Mr 29,895), an extremely toxic protein of the castor bean, inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific adenosine in 23S rRNA.