5: Lecture Features of protein synthesis; posttranslational processing
Today, we will discuss the following: 1. Elongation 2. Termination 3. Fidelity in protein synthesis 4. Energetics of prokaryotic translation 5. Suppressor mutants 6. Eukaryotic translation 1. Antibiotic inhibition of protein synthesis 2. Movie on translation! 3. Regulation of protein synthesis (if time permits)
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Elongation
Three distinct steps to add one amino acid to the growing polypeptide chain. Occurs many times per polypeptide, the number depends on the number of mRNA codons or amino acids in the protein The Elongation Cycle is similar in prokaryotes and eukaryotes. Fast: 15-20 amino acids added per second Accurate: 1 mistake every ~10,000 amino acids
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Schematic of chain elongation in prokaryotes
1. Committing step: EFTu/GTP/AA-tRNA binds to A-site (one phosphate bond broken) 2. Peptide bond formation catalyzed by the 23S rRNA, part of the 50S subunit. The polypeptide chain is transferred from the P-site tRNA to the A-site tRNA. 3. Translocation: movement of peptidyl-tRNA from the Asite to the P-site; and simultaneous transfer of free tRNA from P-site to Esite; movement down one codon driven by EF-G entry into A-site displacing peptidyl-tRNA(one more 3 phosphate bond broken).
Termination
Occurs at stop codons: UAG = Amber UAA = Ochre UGA = Opal/Umber Does not depend on Stop tRNAs. Stop codons bind release factors.
Soluble release factors: RF1 responds to UAA or UAG RF2 responds to UAA or UGA RF3, a GTPase (like EF-Tu and binds in a similar A-site location) RF1/RF2 interact with RF3-GTP, have shapes similar to EF-Tu-GTP-AA-tRNAAA and EF-G, bind to the A-site. Like EF-G, GTP hydrolysis drives the movement of the terminal mRNA codon into the P-site, moving the last tRNA into the E-site and off the ribosome. At the same time, the polypeptide chain is released after hydrolysis of the tRNA-peptide bond. Due to similar structure (molecular mimicry), EF-G and the release factors compete for the A-site. In eukaryotes, only a single release factor, eRF, is necessary. It recognizes all three STOP codons and interacts with GTP.
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Schematic of chain termination
Ribosome stalls when it encounters Stop
Entry of appropriate release factor dimer; RF3 carries GTP to the A-site
Release factor promotes hydrolysis of polypeptide from the peptidyl-tRNA in the P-site GTP hydrolysis causes release of RF-1, RF-3 and the last free tRNA present in the ribosome
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EF-Tu structure in GDP vs. GTP bound state
EF-Tu:GDP (open)
EF-Tu:GtP (compact)
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EF-Tu and kinetic proofreading
What gives rise to specificity in the loading of AA-tRNAs into the A-site? Energy of one GTP bond. How? Kinetic proofreading: there are many EF-Tu:GTP:AA-tRNA (ternary) complexes in the cell. But, for a particular ribosome sitting on a particular codon, we need the right AA-tRNA. If codon and anticodon are not complementary, the ternary complex comes right out of the A-site. If the codon and anticodon base-pair, interaction is stabilized resulting in increased time in the A-site, breakdown of the GTP bond, and then release of EF-Tu: GDP (EF-Tu: GDP cant bind to tRNA and doesnt fit in the Asite). Also, induced fit caused by base-pairing squeezes the ternary complex down into the A-site, causing hydrolysis of GTP. If the fit is right, the AA-tRNA is locked in the A-site.
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Molecular mimicry of EF-G and EF-Tu:GTP:AA-tRNA
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Coupled transcription and translation in prokaryotes
At 37oC, rate of translation in E. coli = 15 AA/sec. Rate of transcription is ~45 nucleotides/sec. Transcription and translation are coupled! (Not so in eukaryotes because these processes occur in different places)
Dr. Hamkalos demonstration that transcription and translation are coupled in E. coli.
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Energetics of prokaryotic protein synthesis
Energy cost for synthesis of a prokaryotic protein with N ami no acids: 2N ATPs to charge tRNA (ATP -> AMP + PP -> AMP + 2Pi) 1 GTP for initiation (IF2) N-1 GTPs to position tRNA for N-1 peptide bonds (EF-Tu) N-1 GTPs for N-1 translocation steps (EF-G) 1 GTP for termination (RF-3) ==== 4N A total of 4 high energy phosphate bonds is cleaved per AA. Each ATP or GTP generates ~40 kJ/mol. Each peptide bond costs ~160 kJ/mol in the cell, yet an uncatalyzed chemical reaction to form a peptide bond costs only ~20 kJ/mol. Why is it so costly to make a peptide bond on a ribosome? T he excess energy is used for generating an accurate, defined polypeptide seque nce, not a random one or a combination of multiple possibilities. As stated abo ve, the fidelity is due to two mechanisms:
1. Proofreading before aminoacyl adenylate intermediate is attached to tRNA. 2. Kinetic proofreading before peptide bond formation. Each major step in protein synthesis, except peptide bond formation itself, involves hydrolysis of GTP to GDP.
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Nonsense suppression and suppressor tRNAs
Stop codons:
UAG Amber UAA Ochre UGA Opal/Umber
Originally defined Amber, Ochre and Opal suppressors as E. coli mutants that would allow T4 phage rII locus nonsense mutants to grow.
Crick and Brenner used these mutants to discover the Stop codons. But how do these nonsense suppressor mutants work? Stop codons dont bind to AA-tRNAs in the A-site, they bind to release factors (RFs). Suppressor mutants are due to mutations in tRNA genes, in the anticodon region. They mask the Stop codons and prevent premature termination (of the nonsense mutants).
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Examples of nonsense suppressor tRNAs
Mutation in the anticodon of a tRNA so that it can recognize a Stop codon (e.g. Amber). Example: mutation of uncommon (recall codon bias) TyrtRNATyr. Mutation in the anticodon does not alter recognition by the cognate tRNA synthetase. Tyr is inserted at Amber Stop codons.
AA-tRNA tRNATyr Sup-tRNATyr Anticodon GUA CUA Result Inserts Tyr at UAC codon Inserts Tyr at UAG codon
Another example: mutation in the anticodon of an uncommon Trp tRNA gene so that it will base-pair with Amber Stop codons. But Trp AA-tRNA synthetase doesnt recognize this mutant tRNA. It is recognized by GlntRNA synthetase which adds glutamine to this mutant tRNA. Gln is added at Amber Stop codons.
AA-tRNA tRNATrp Sup-tRNATrp Anticodon CAA CUA Result Inserts Trp at UUG codon Inserts Gln at UAG codon
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Translation in Eukaryotes
In prokaryotes, we have a Shine-Dalgarno sequence to align the ribosome onto the correct AUG codon. Dont have S-D sequences in eukaryotes. But, we have a 5 7-Methyl GTP cap, poly-A & Kozak sequence:
Both 7-Methyl GTP cap and poly-A tail are added post-transcriptionally. Both the cap and the tail are involved in orienting the mRNA, and the cap is important for orienting the ribosome on the correct AUG codon. 13
5 TOP sequence in some eukaryotic mRNAs
About 100-200 eukaryotic mRNAs have a 5 Terminal Oligo-Pyrimidine sequence rather than a 5 7-Methyl GTP cap. All ribosomal protein mRNAs have 5 TOP sequence as do some of the elongation factor mRNAs. Very important for translation of proteins involved in translation and cell growth. TOP sequence is found in very short 5 untranslated regions (UTR), a stretch of 6-12 pyrimidines. In quiescent cells, only 20-30% of TOP-containing mRNAs are found on polysomes, but in activated cells, 70-80% of these are found on polysomes. this So, is a regulated form of translation of a specific subset of mRNAs! Dont know much about exactly how ribosome recognizes TOP sequences, but involves phosphorylated S6 (small subunit) protein. Specialized 14 proteins involved in cell growth!
Overview of eukaryotic initiation
Eukaryotic initiation occurs in three steps. Numerous initiation factors, recognize the 5 7-Methyl GTP cap. There are many steps in which translational regulation occurs (more on this next time).
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Eukaryotic initiation Step 1: 43S pre-initiation complex formation
Association of Met-tRNAiMet with eIF2. eIF2 is charged with GTP by eIF2B. This is the ternary complex: Met-tRNAiMet-eIF2-GTP. eIF1A and eIF3 release the 60S subunit from a free assembled ribosome by associating with the 40S subunit. The ternary complex binds to the eIF1A/eIF3/40S subunit to form the 43S preinitiation complex. Note: mRNA is not bound at this stage and does not direct the formation of the 43S preinitiation complex. Distinct from the formation of the prokaryotic 30S preinitiation complex, because there is no Shine-Dalgarno sequence.
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eIF2 charging: the eIF2B cycle
eIF2 is charged with GTP by eIF2B eIF2B is a GTP exchange factor, thus it exchanges GTP for GDP on eIF2. To recycle charged eIF2B, it is itself phosphorylated by a kinase that exchanges a high energy phosphate on ATP. Well come back to this next time in our discussion of translational regulation.
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Eukaryotic initiation, Step 2: formation of the 48S preinitiation complex
This step accomplishes binding and orientation of the mRNA to the preinitiation complex. It does so by the formation of a cap-binding complex (AKA eIF4F).
eIF4F = eIF4A + eIF4E + eIF4G. eIF4E activity is blocked by 4E-BP (eIF4E binding protein). eIF4F binds first to the 5 cap, then poly-A binding protein (Pab1p) binds to the poly-A tail and to eIF4G, which acts as a scaffold to bind the 40S subunit. eIF4F has helicase activity, so in addition to promoting assembly of the 48S preinitiation complex, it unwinds the mRNA to promote efficient translation (RNAs often have complex 2o and 3o 18 structures).
Eukaryotic initiation, Step 3: formation of the 80S initiation complex
Achieved by binding of the 60S ribosomal subunit with the 48S preinitiation complex composed of the 40S subunit and the mRNA positioned at the correct initiator codon. Transient scanning of the mRNA on the 48S pre-initiation complex occurs in search for AUG codon (usually 40-150 bases 3 of the cap). Translation doesnt necessarily start at the first AUG encountered; the pre-initiation complex searches for the best AUG (closest to the Kozak consensus). Once the correct AUG is found, GTP is hydrolyzed from the ternary complex (eIF2:GTP:Met-tRNAiMet). This causes release of all of the initiation factors and tight binding of the 40S and 60S subunits. In this state, the initiation complex has the mRNA situated appropriately with the MettRNAiMet in the P-site and the A-site awaiting elongation.
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Eukaryotic translation: elongation
Very similar to elongation in prokaryotes. Two elongation factors involved: EF1 = EF1A + EF1B (EF1B and EF1B ) EF2 EF1A works like EF-Tu. Binds GTP and shuttles AA-tRNAs to the A-site EF1B works like EF-Ts. Exchange factor for charging EF1A with GTP. EF2 works and looks like EF-G: charged with GTP, involved in ribosomal translocation
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Eukaryotic translation: termination
Only one release factor in eukaryotes, comprised of two 55 kDa subunits eRF binds the A-site of the ribosome in a GTP-dependent manner. Upon binding, promotes: 1. Hydrolysis of peptide bond on the tRNA in the P-site 2. Release of deacylated tRNA 3. mRNA release Process requires energy provided by GTP bound to eRF.
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Antibiotic inhibition of protein synthesis
Antibiotics: inhibitors of cell growth (cytostatic) or cell survival (cytotoxic). Derived from fungi or bacteria (they likely produce it to combat competitors for limited resources in nature).
Many antibiotics target protein synthesis because it is so crucial to cell growth and survival. In many cases, they selectively block either prokaryotic or eukaryotic translation.
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Prokaryotic inhibitors
Streptomycin: affects 30S function At high [strep], is cytotoxic because it poisons 70S preinitiation complexes At low [strep], is cytostatic; induces misreading of codons. Cells not dead, but lots of non-functional proteins. Puromycin: Binds to A-site (both prokaryotic and eukaryotic). Looks like AA-tRNAAA, and causes aberrant peptidyl-transferase reaction. Results in premature chain termination.
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Eukaryotic protein synthesis inhibitors
Puromycin Cycloheximide: inhibits peptidyl transferase on 60S subunit. Cytotoxic or cytostatic. Diptheria Toxin: inactivates eEF2 via ADP ribosylation. Very few molecules can kill cells! Ricin: produced by castor bean Ricinus communis. Two chains: B chain allows for cell entry, A chain is the toxin. Enzymatic activity attacks the 28S rRNA (in the 60S subunit), thereby eliminating peptidyl-transferase activity. A single A-chain molecule can inactivate 50,000 ribosomes!
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Antibiotic resistance genes
Note that many unicellular organisms have antibiotic resistance genes. One example is the hygromycin resistance gene hphr. Hyg is an aminoglycosidic antibiotic produced by Streptomyces hygroscopicus. The hphr gene is a kinase and inactivates the antibiotic by phosphorylating it.
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Translational regulation
Prokaryotes: 1) Shine-Dalgarno sequence (100X change in translation rate) 2) Attenuation Eukaryotes: 1) mRNA masking, antisense and RNAi 2) Regulation of initiation In eukaryotes, there are many places in which translation can be regulated
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mRNA and translational regulation
1. mRNA masking: mRNA binding proteins prevent initiation. Major form of regulation during early embryonic development. 2. Antisense RNA: short segment of RNA, complementary to mRNA, that forms double stranded RNA (dsRNA) which cannot be translated by ribosome. 3. RNAi: RNA interference or gene silencing. Short RNA molecules bind to mRNA, create short stretches of dsRNA, activates special RNAases. We can mimic RNAi effect in the lab by introduction of short synthetic RNA into cells. siRNA base-pairs with target mRNA, causing its destruction. May also be useful therapeutically.
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Inactivation of eIF2 via phosphorylation
Phosphorylation of eIF2 by eIF2a kinase inhibits translation; Phospho-eIF2 binds too tightly to eIF2B (the charging partner) which is limiting in cells. Cells run out of eIF2B for charging eIF2 with GTP, so translation initiation halts.
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Growth factor regulation of translation
GF signaling activates mTOR 1. mTOR phosphorylates 4E-BP. This frees eIF4E and allows translation to initiate. 2. mTOR activates S6 kinase, leading to the phosphorylation of ribosomal protein S6. Phospho-S6 promotes translation of 5 TOP containing mRNAs.
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