Ch 24, Exam 4 - Biochemistry and Molecular Biology of the Gene - UNT BIOC 4570

2. Terms: a. AUG b. UAA, UAG, UGA c. Releasing factors d. Ribosome binding site

A) 679: The signal of initiating a polypeptide chain is a special initiation codon that marks the start of the reading frame. usually the initiation codon is the triplet AUG, but in bacteria GUG or UUG may also be used. An mRNA contains many AUG triplets, so how is the corret intitation codon recognized as providing the starting point for translation? The sites on mRNA where translation is initiated can be identified by binding the ribosome to mRNA under conditions that block the elongation so that the ribosome remains that the initiation site. When ribonuclease is added to th blocked intitiation complx, all the regions of mRNA outside the ribosome are degraded, but those actually bound to it are proteted, as illustrated in figure 24.12. THe protected fragments can then be recovered and characterized. The intitiation sequences protected by bacterial ribosomes are ~30 bases long. The ribosome binding sites of different bacterial mRNAs displa two comon features: The AUG or less often GUG or UUG initiation codon is always included within the protected sequence. Approximately 10 bases upstream of the AUG is a sequence that corresponds to part or all of the hexamer: 5'...AGGAGG...3'. This polypurine stretch is known as the shine dalgarno sequence. It is complementary to a highly conserved sequence close to the 3' end of 16S rRNA. B)Pg 692: Three codons terminate translation. THe codons UAA (ochre), UAG (Amber), and UGA (opal) terminate translation. In bacteria they are most often used with relative frequencies UAA>UGA>UAG Only 61 nucleotide triplets specify amino acids. The other three triplets are termination codons (also known as nonsense codons, or stop codons), which end translation. They have casual names from the history of their discovery. The UAG triplet is called the amber codon, UAA is the ochre codon, and UGA is the opal codon. The nature of these triplets was originally shown by a genetic test that distinguished two types of point mutations. A point mutation tha changes a codon to represent a diffferent amino acid is called a missense mutations. One amino acid replaces the other in the protein, the effect on protein function depends on the site of mutatino and the nature of the amino acid replacement. C) Releasing factors: pg 693: Termination codons are recognized by protein release factors, not by aminoacyl tRNAs. The structure of the class 1 release factors (RF1 and RF2 in e coli) resemble aminoacyl tRNA EF tu and EF G. The class 1 release factors respond to specific termination codons and hydrolyze the polypeptide tRNA linkage. The class 1 release factors are assisted by class 2 release factors (such as RF3) that depend on GTP. The mechanism is similar in bacter (which have two types of class 1 release factors) and eukaryotes, which only have one class 1 release factor. Two stages are involved inending trnaslation. TH termination reaction itself involves release of the protein chain from the last tRNA. THe posttermination reacction involes the release of the tRNA and mRNA and dissociatition of the ribosome into its subunits. None of the terminatino codons is represented by a tRNA. They functino in an entirely different manner from other codons and are recognized directly by protein factors. The reaction does not depend on codon anticodon recognition, so there seems to be no particular reason why it should require a triplet sequence. resumbly this reflects the evolution of the genetic code. Termination codons are recognized by class I release factors (RF). in E coli, two class 1 release factors are specific for different sequences. RF1 recognizes UAA and UAG, rf2 recognizes UGA and UAA. The factors at the ribosomal A site and require polypeptidyl tRNA in the P site. The RFs are present at much lower levels that initiation or elongation factors; there are ~600 molecules of each per cell, equivalent to one RF per 10 ribosomes. At one time there was probably only a single release factor that recognized all termination codons, which later evolved into two factors with specificities for particular odons. In eukaryotes, there is only a single class I release factor, called eRF. THe efficiency with which the bacterial factors recognize their target codons is influenced by the bases on the 3' side. The class 1 release factors are assisted by class 2 release factors, which are not codon specific. THe class 2 factors are GTP binding proteins. in E coli, the role of the class 2 factor, RF3, is to release the class 1 factor from the ribosome. RF3 is a GTP binding protein that is related to the elongation factors. d. Ribosome binding site: 677: Bacterial ribosomes engaed in elongating a polypeptide chain exist as 70S particles. At termination, they are released from the mRNA as free ribosomes or ribosomal subunits. In growing bacteria, the majority of ribosomes are synthesizing polypeptides; the free pool is likely to contain ~20% of the ribosomes. Ribosomes in the free pool can dissociate into separate subunits; this means that 70S ribosomes are dynamic equilibrium with 30S and 50S subunits. Intiation of translation is not a function of intact ribosomes, but is undertaken by the separate subunits, which reassociate during the initiation reaction. Figure 24.9 summarizes the ribosomal subunit cycle during translation in bacteria. Initiation occurs at a special sequence on mRNA called the ribosome-binding site (including the shine dalgarno sequence) This is a short sequence of bases that precedes the coding region and is complementary to a portion of the 16S rRNA. The small and lage subunits associate at the ribosomal binding site to form an intact ribosome. The reaction occurs in two steps: recognition of mRNA occurs when a small subunit binds to form an initiation complex at the ribosome binding site. A large subunit then joins the complex to generate a complete ribosome. Although 30S subunit is involved in initiatino, it is not sufficient by itself to undertake the reactions of binding mRNA and tRNA; this requires additional proteins called initiation factors.

1. What are the 3 tRNA binding sites found in a ribosome and what does each do?

Pg 674: The ribosome has three tRNA binding sites. AN aminoacyl-tRNA enters the A site. Peptidyl-tRNA is bound in the P site. Deacylated tRNA exits via the E site. An amino acid is added to the polypeptide chain by transferring the polypeptide from the peptidyl-tRNA in the p site to aminoacyl-tRNA in the A site. An amino acid is brought to the ribosome by an aminoacyl-tRNa. Its addition to the growing polypeptide chain occurs by an interaction with the tRNA that bought the previous amino acid. Each of these tRNAs lies in a distinct site on the ribosome. Figure 24.3 shows that the two sites have different features. An incoming aminoacyl-tRNA binds to the A site. Prior to the entry of aminoacyltrna the site exposes the codon representing the next amino acid to be added to the chain. THe codon representing the most recent amino acid to have been added to the nascent polypeptide chain lies in the p site. THis site is occupied by peptidyl-tRNA, a tRNA carrying th nascent polypeptide chain. Figure 24.4 shows that the aminoacyl end of the tRNA is located on the large subunit, whereas the anticodon at the other end interacts ith the mRNA bound by the small subunit. So the P and A sites each extend across both ribosomoal subunits. For a ribosome to form a peptide bond, it must be in the state shown in step one in figure 24.3, when a peptidyl tRNA is in the P site and aminoacyl tRNA is in the A site. Peptide bond formation cocurs when the polypeptide carried by the peptidyl tRNA is transferred to the amino acid carried by the aminoacyl tRNA. This step requires correct positioning of the aminoacl ends of the two tRNAs within the large subunit. THis reaction is catalyzed by the large subunit of the ribosome. transfer of the polypeptide generates the ribosome shown in step 2 of figure 24.3 in which the deacylated tRNA lacking any amino acid, lies in the p site, and a new peptidyl t RNA is in the A site. THis peptidyl tRNA is one amino acid residue longer than the peptidyl tRNA that had een in the P site in step 1. The ribosome now moves one triplet along the messenger RNA. this stage is called translocatino. THe movement transfers the deacylated tRNA out of hte P site and moves the peptidyl tRNA into the p site. The next codon to be translated now lies in the A site, ready for a new aminoacyl tRNA to enter, when the cycle will be repeated. The deacylated tRNA leaves the ribosome via another tRNA binding site, the E site. THis site is transiently occuped by the tRNA en route etween leaving the P site and being released from the ribosome into the cytosol. Thus, the flow of the tRNA is into the A site, through the P site, and out through the E site.

5. What is a Shine-Dalgarno Sequence (location, properties and function). Why are these necessary in prokaryotic genomes/mRNAs but not in/on eukaryotic ones?

Pg 679: The initiation sequences protected by bacerial ribosomes are ~30 bases long. The ribosome binding sites of different bacterial mRNAs display two common features: the AUG or less often, GUG or UUG initiation codon is awlays incldued within the protected sequence. Approximately 10 bases upstream of the AUG is a sequence that corresponds to apart or all of the hexamer. 5'....AGGAGG...3' This polypurine stretch is known as cthe shine dalgarno sequence. it is complementary to a highly conserved sequence close to the 3' end of 16S rRNA. The extent of complementarity differes with individual mRNAs and may extend from a four base core sequence GAGG toa nine base sequence extending beyond each end of the hexamer. Writen in reverse direction, the rRNA sequence is the hexamer UCCUCC...5'.

9. How many different methionine tRNAs (reading codon AUG) must an E. coli cell have and why? How does the methionine amino acid incorporated as the first amino acid of a protein differ from internal methionines in an E. coli cell? What would be the correct answer to these questions for human cells?

Pg 680: Synthesis of all polypeptides starts with the same amino acid: methionine.tRNAs recognizeing the AUG codon carry methionine, and two type s of tRNA can carry this amino acid. One is used for initiation, the other for recognizing AUG codons during elongatino. In bacteria, mitochondria, and chloroplasts,the intiator tRNA carrries a methinoine residue that has been formylated on its amino group, forming a molecule of n-formyl-methionyl tRNA. the tRNA is known as tRNAfmet. The name of the aminoacyl tRNA is usually abbeviated to fmet-tRNA. The initator tRNA gains its modified amino acid in a two stage reaction. First, it is chargedwith the amino acid to generate met-tRNA, and then the formylation reaction shown in figure 24.14 blocks the free nh2 group. Although the blocked amino acid group would later prevent the initiator from participating in chain elongation, it does not itnerfere with the ability to initiate a polypeptide. This tRNA is used only for initiation. It reocnizes the codons AUG or GUG (occasionally UUG). The codons are not reocnizes qually wel; the extent of initiation decliens by about half when AUG is replaed by GUG and dclines by about half again when UUG is emplyoed.

7. Explain how diphtheria toxin acts to inhibit eukaryotic protein synthesis.

Pg 692 Translocation is an intrinsic property of the ribosome that requires amajor change in structure. This intrinsic translocation is activated by EF-G in conjunctino with GTp hydrolysis, which occurs before translocatino and accelerates the ribosomal movement. Te most likely mechanism is hat GTp hydrolysis causes a change in the structure of EF-G, which in turn forces a change in the ribosome structure. An extensive reoritentation of EF-G occurs at translocation. Before tranlocation, it is bound across the two bribosomal subunits. Most of its contacts with the 30S subunit are made by a region called domain 4, which is inserted into the A site. This domain could e responsible for displacing the tRNA. Afer translocation, domain 4 is instead oriented towards the 50S subunit. The eukaryotic counterpart to EF-G is the protein eEF2, which functions in a similar manner to a translocase dependent on GTP hydrolysisis. Its action also is inhiited by fusidic acid. A stable complex of eEF2 with GTP can be islolated and the complex can bind to ribosomes with consequence hydrolysis of its GTP. A unique reaction of eEF2 is its suscptibility to dihteria toxin. The toxin uses nicotinamide adenine dinucleotide NAD as a cofactor to transfer an adensoine diphosphate ribosyl (ADPR) moiety onto the eEF2. The ADPR-eEF2 conjugate is inactie in translation. The substrate for the attachment is an unusual amino acid that is produced by modifying a histidine ; it is ocmmon to the eEF2 of many species. the ADP ribosylation is responsible for the lethal efects of diphtheria toxin. THe reaction is extermely effective: a single molecule of toxin can modify enough eEF2 molecules to kill a cell.

8. Describe/diagram the reaction catalyzed by peptidyl transferase. Where is this activity found and what is its physical nature?

Pg 702: 23S rRNA has peptidyl transferase activity. Peptidyl transferase activity resides exlcusively in the 23s rRNA. The sites involved in the functions of 23s rRNA are less well identified than those of 16S rRNA, but the same general pattern is oersved. Bases at certain positinos affect specific functions, bases at some positinos in 23S rRNA are affected by the conformation of the A site or the P site. In particular, oligonucleotides derived from the 3' CCA terminus of tRNA protect a set of bases in 23S rRNA that essentially are the same as those protected by peptidyl tRNA. This suggests that the major interaction of 23s rRNA with peptidyl tRNA in the p site involevs the 3' end of the tRNA. THe tRNA makes contact with the 2s rRNA in both the P and A sites. at the P site, G2552 of the 23S rRNA base pairs with C74 of the peptidyl tRNA. A Mutation in the G in the rRNA prevents interaction with tRNA, but interaction is restored by a compensating mutatino in the C of the amino aacceptor end of the tRNA. At the A site, G2553 of the 23s rRNA base pairs with C75 of the aminoacyl tRNA. THus, there is a close role for rRNA in both the tRNA binding sites. as structure studies continue to emerge, the movements of tRNA between A and P sites in terms of making and breaking contacts with rRNA will be elucidated. ANother site that binds tRNA is the E site, which is localized almost exlcusively on the 50S subunit. Bases affected by its conformatino can be identified in 23S rRNA. What is the nature of the site on the 50S subunit that provides peptidyl transferase function? A long search for ribosomal proteins that might possess the catalytic activity was unsuccessfull and led to the discovery that the ribosomal RNA of the large subunit can catalyize th formation of a peptide bond between peptidyl tRNa and aminoacyl tRNA. The inveolemnt of rRNA was first indicated because a region of 23s rRNA is the site of mutations that confer resistance to antibiotics that inhibit peptidyl transferase.

6. What is the site of action of puromycin? Why are many compounds of this type useful medically as antibiotics? Why can chronic use of antibiotics be harmful to humans?

The ribosome remains in place while the polypeptide chain is enlongated by transferring the polypeptide attached to the tRNA in the P site to the aminoacyl tRNA in the A site. the reaction is shown in figure 24.26. The activity responsible for synthesis of the pepetide bond is called peptidyl transferase. It is a functino of the large (50S or 60S) ribosomal subunit. The reaction is triggered when EF-Tu releases the aminoacyl end of its tRNA, which then swings into a location close to the end of the peptidyl tRNA. THis site has a peptidyl transferase activity that essentially ensures a rapid transfer of the peptide chain to the aminoacyl tRNA. BOth rRNA and the 50S subunit proteins are necessary for this actiity, but the actual act of catylsis is aproperty of the ribosomal RNA of the 50S subunit. The nature of the transfer reaction is revealed by the ability of the antibiotic puomycin to inhibit translation. Puromycin resembles an amino acid attached to the terminal adenosine of tRNA. Figure 24.27 shows that puromycin has an N instead of the O that joins an amino acid to a tRNA. The antibiotic is treated by the ribosome as though it were an incoming aminoacyltRNA, after which the polypeptide attached tot the peptidyl-tRNA is transferred to the NH2 group of puromycin. THe puromycin moeiety is not anchored to the A site of the ribosome; as a result, the polypeptidyl puromycin adduct is releasedf from the ribosome in the form of polypeptidyl puromycin. THis premature termination of translation is responsible for the lethal action of the antibiotic.

4. Describe the cycle by which EFTu and EFTs act to deliver charged tRNAs to an E. coli ribosome. Use diagrams for clarity

pg 690: Translocation requires EF-G, whsoe structure resembles the aminoacyl-tRNA-EFTu-GTP complex. Binding of EF-Tu and EF-G to the ribosome is mutually exclusive. Translocation requires GTP hydrolysis, which triggers a change in EF-G,which in turn triggers a change in ribosome structure. Translocation requires GTP and another elongation factor, EF-G (the eukaryotic homolog of EF-G is eEF2). This factor is a major consitutent of the cell; it is rpesent at a level of ~1 copy per ribosome (0,000 molecules per cell). Ribosomes cannot bind EF-Tu and EF-G siultaneously, so translation follows the cycle illustrated in figure 24.30, in which the factors are alternatively bound to and released from the ribosome. Thus, EF-Tu-GDP must be relseased before EF-G can bind, and then EF-G must be released before aminoacyl-tRNA-EF-Tu-GTP can bind. Does the ability of each elongation factor to excluse the other rely on an allosteric effect on the overall conformation of the ribosome or on diret competititino for overlapping binding sites? Figure 24.31 shows an extraordinary similarity between the structures of the tertiary complex of aminoacyltRNA EF tu GDP and EF G. The strucutre of EF-G mimics the overal structuer of Ef-Tu bound to the amino acceptor stem of aminoacyltRNA. This suggests that they compete for the same binding site (presumbably in the vicinity of the A site). The need for each factor to be released before the other can bind ensures that the events of translation proceed in an orderly manner. BOth elongation factors are monomeric GTP binding proteins that are active hen bound to GTP but inactive when bound to GDP. The triphosphate form is required for binding to the ribosome, which ensures that each factor obtains access to the ribosome only in the company of the GTP that it needs to fulfill its function. EF-G binds to the ribosome to facillitate translocation and then is released following ribosome movement. Ef-G can still bind to the ribosome when GMP-PCP is sibstituted for GTp, so the presence of a guanine nucleotide is needed for binding, but its hydrolysis is not absolutely essential for translocation (although translocation is much slower in the absence of GTP hydrolysis). The hydrolysis of GTP is needed to release EF-G. THe need for EF-G release was discovered by the effects of the steroid antibiotic fusidic acid, which jams the ribosome in its post translocation state.In the presence of fusidic acid, one round of translocatino occurs: EF-G binds to the ribosome, GTP is hydrolyzed, and the ribosome muves three nucleotides. However, fusidic acid stabilizes the ribosome EF G GDP complex so that EF g and GDP remain on the ribosome instead off being released. As ar esult, the ribosome cannot bind aminoacyl tRNA and no further amino acids can be added to the chain. Translocation is an intrinsic property of the ribosome that requires a major change in structure. This intrinsic translocation is activated by EF-G in conjunction with GTP hydrolysis, which occurs before the translocation and accelerates the ribosome movement. The msot likely mechanism is that GTP hydrolysis causes a change in the structure of EF-G, which in turn forces a change in the ribosome structure. An extensive reorientation of EF-G occurs at translocation. Before translocation, it is bound acros the two ribosomal subunits. Most of its contacts with the 30S subunit are madee by a region called domain 4, which is inserted into the A site. This domain could be responsible for displacing the tRNA. After translocation, domain 4 instead oriented towards the 50S subunit. THe Actually, I think 24.10, on page 687: EF-Tu is a monomeric G protein whose active form bound to GTP binds to aminoacyl tRNA. the EF tu GTP aminoacyl tRNA complex binds to the ribosomes A site. There's a diagram on page 687. Once the complete ribosome is formed at the initation codon, the stage is set for a cycle in which an aminoacyl tRNA enters the A site of ar ibosome whose P site is occupied by a peptidyl tRNA. Any aminoacyl tRNA except the initiator can enter the A site. Its enry is mediated by an elongation factor (EFTu) in bacteria. The process is similar in eukaryotes. Ef-Tu is a highly conserved protein throughout bacteria and mitochondria and is homologous to its eukarotic counterpart. Just like its counterpart in itiation (IF-2), EF-Tu is associated with the ribosome only during the process of aminoacyl-tRNA entry. once the aminoacyl tRNA is in place, EF tu leaves the ribosome to work agian with another aminoacyl tRNA. Thus, it displays the cyclic association with, and dissociation from, the ribosome that is the hallmark of the accessory factors. 24.25 depicts the role of EF-Tu in bringing aminoacyl tRNA to the A site. EF-tu is a monomeric GTP binding protein that is active when bound to GTP and inactive when bound to GDP. The binary complex of EF-Tu-GTP binds aminoacyl tRNA to form a ternary complex oc aminoacyl-tRNA EF tu GTP. THe ternary complex binds only to the A site of ribosomes hose P site is already occupied by peptidyl tRNA. This is the critical reaction in ensuring that the aminoacyl tRNA and peptidyl tRNA are correctly positioned for peptide bond formation. Aminoacyl tRNA is loaded into the A site in two stages. First, the anticodon end binds to the A site of the 30S subunit. Then, codon antiocodon recognition triggers a change in the conformatino of the ribosome. This stabilizes the tRNA binding and causes EF-Tu to hydrolize its GTP. The CCA end of the tRNA now moves into the A site on the 50S subunit. The binary complex EFTu-GDP is released. THis form of EF-Tu is inactive and does not bind aminoacyl-tRNA effectively. The guanine nucleotide excchange factor, EF-Ts, mediates the regeneration of the used form, Ef-Tu-GDP, into the active form EF-Tu-GTP. First, EF-Ts displaces the GDP from Ef-Tu, forming the combined factor EF-Tu-EFTs . Then the Ef-Ts is in turn displaced by GTP, reforming the Ef-Tu-GTP. The active binary complex binds aminoacyltRNA and the relased EF-Ts can recycle. There are ~70,0000 molecules of Ef-TU per bacterium, five percent of the total bacterial protein, which approaches the number of aminoacyl-tRNA molecules. THis implies that most aminoacyl-TRNAs are likely to be present in ternary complexes. There are only ~10,000 molecules of EF-Ts per cell, about the same as the number of ribosomes. The kinetics of the interactino between EF-Tu and EFTs suggest that the EF-Tu-EF-Ts complex exists only transiently, so that the EFTu is very rapidly converted to the GTP bound form, and then to a ternary complex.

10. What is an IRES with regard to eukaryotic translation? What is an example of where they are required?

pg 684: The majority of eukaryotic initiation events inolve scnaning from the 5' cap, bu there is an alternative means of initiation, used especially by certain viral RNAs,in which a 40S subuninit associates directly with an internal site call an internal ribosome entry site (IRES). In this case, any AUG codons that may be in the 5' untranslated region are bypassed entirely. THere are few sequence homologies between known IRES elements, WE can distinguish three tpyes on the basis of their interaction with the 40S subuninit. THe most common type of IReS includes the AUG intitiation codon at its upstream boundary. THe 40S subunit binds directly to it, using a subset of the same factors that are required for intitatino at 5' ends. Another type is located as much as 100 nucleotides upstream of the AUG, requiring a 40S subunit to migrate, again probably by a scanning mechanism. An eceptional type of IRES in hepatitis C virus can bind a 40S subunit directly, without requiring any intitation factors. The order of events is different form all other ekaryotic intitiation. Following 40S mRNA binding,a complex containing intitiator factors and the intitiator tRNA binds.

3. Describe the process of protein synthesis in an E. coli cell. Include all protein factors, energy sources, etc. Use diagrams to aid in clarity.

pg 695 is as close as i can find for something like this. http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.mun.ca/biochem/courses/3107/Lectures/Topics/Nine_steps.html That's not bad