Chapter 12: Translation
Components needed for translation
1. *ribosomes (rRNA + protein)* - sites of protein synthesis 2. mRNA - determines order of amino acids in protein chain, message to be translated, bound to ribosome 3. tRNA - responsible for translating message 4. other protein factors
Steps of translation or protein biosynthesis
1. Amino acid activation - amino acid covalently bonded to tRNA, involves tRNA and aminoacyl-tRNA synthetases. 2. Initiation - formation of initiation complex, the binding of the first aminoacyl-tRNA to the start site on the ribosome. polypeptide chains formed (1 of 3 steps) 3. Elongation - formation of peptide bonds between successive amino acid residues. (repeated many times until polypeptide chain is complete) 4. Termination - release of newly formed protein from ribosome
Process of chain initiation in eukaryotes
1. Assembly of 43S preinitiation complex - met-tRNAi complexed to eIF2 binds small subunit - Other eIF's bound 2. Recruitment of mRNA - aided by 5' cap. - order is different than that of prokaryotes --- mRNA binds before initiator tRNA in that case 3. Recruitment of 60S subunit - 80S initiation complex formed, GTP hydrolyzed, initiation factors released
Steps in forming 70S initiation complex
50S binds GTP hydrolyzed IF's released
Components of elongation
70S complex, GTP, EF-Tu (temp. unstable), and EF-Ts (temp. stable)
Amino acid activation is catalyzed by
aminoacyl-tRNA synthetases
A genetic code in which two bases encode a single amino acid is not adequate for protein synthesis. Give a reason why.
A code in which two bases code for a single amino acid allows for only 16 possible codons (4^2), which is not adequate to code for 20 amino acids. With three bases, there are 4^3 possibilities, or possible 64 codons, which is more than enough to encode the 20 amino acids.
Universal start codon
AUG (methionine)
How would protein synthesis be affected if a single codon could specify the incorporation of more than one amino acid (an ambiguous code)?
An ambiguous code would allow for variation in the amino acid sequence of proteins. Consequently, there would be variation in function, including a number of nonfunctional proteins.
What is the role of the 50S ribosomal subunit in prokaryotic protein synthesis?
Attachment of the 50S ribosomal subunit to the 30S subunit in the initiation complex is needed for protein synthesis to proceed to the elongation phase.
How does it improve the efficiency of protein synthesis to have several binding sites for tRNA close to each other on the ribosome?
Because the tRNAs are bound in proximity to each other on the ribosome, the growing polypeptide chain and the amino acid to be added are also close to each other. This facilitates formation of the next peptide bond.
In prokaryotic protein synthesis, N-formylmethionine (fmet) is the first amino acid incorporated, whereas (normal) methionine is incorporated in eukaryotes. The same codon (AUG) serves both. What prevents methionine from being inserted into the beginning and N-formylmethionine in the interior?
Different tRNAs and different factors are involved. Initiation requires IF-2, which recognizes fmet-tRNAfmet but not met-tRNAfmet. Conversely, in elongation, EF-Tu recognizes met-tRNAmet but not fmet-tRNAfmet.
Steps of elongation
EF-Tu bound to 2nd aminoacyl-tRNA and GTP EF-Tu delivers tRNA to A site Anticodon of tRNA base pairs with codon of mRNA Hydrolysis of GTP releases EF-Tu EF-Ts regenerates EF-Tu-GTP Transpeptidation Translocation
How does eukaryotic mRNA differ from that of prokaryotes
Eukaryotic mRNA has 5' cap and 3' PolyA tail
Second genetic code
Extra level of proofreading by aminoacyl-tRNA synthetases. Contributes to accuracy of process Need to pair right amino acid with right tRNA
Steps of amino acid activation
First, the amino acid forms a covalent bond to an adenine nucleotide, producing an aminoacyl-AMP. The free energy of hydrolysis of ATP provides energy for bond formation. The aminoacyl moiety is then transferred to tRNA, forming an aminoacyl-tRNA. 2. Ester linkage formed between carboxyl of amino acid and -OH of ribose of 3' end of tRNA.
What is the energy cost per amino acid in prokaryotic protein synthesis? Relate this to low entropy.
Four high-energy phosphate bonds per amino acid: two in aminoacyl-tRNA formation, one in elongation with EF-Tu, and one in translocation from the A to the P site, involving EF-G. Forming a peptide bond requires about 5 kcal/mol. This is an expenditure of about 30 kcal/mol peptide bonds. This is the price of low entropy and high fidelity.
Why doesn't GlyRS need a proofreading domain?
Gly is the smallest amino acid, so the aminoacylation site in GlyRS can be small enough to prevent the entry of any other amino acid
What sequence in the prokaryotic release factors is important to the hydrolysis of the peptide bond?
Gly-Gly-Gln
Wobble base of anticodon ___-bonds to ____ of codon
H, third base
The amino acid hydroxyproline is found in collagen. There is no codon for hydroxyproline. Explain the occurrence of this amino acid in a common protein.
Hydroxyproline is formed from proline, an amino acid for which there are four codons, by posttranslational modification of the collagen precursor.
The nucleoside inosine frequently occurs as the third base in codons. What role does inosine play in wobble base pairing?
Hypoxanthine is the most versatile of the wobble bases; it can base pair with adenine, cytosine, or uracil.
Steps in forming the 30S initiation complex
IF3 bound to 30S ribosomal subunit - facilitates binding of mRNA - Prevents premature binding of 50S IF2 - binds initiator tRNA and escorts it to 30S and escorts tRNA to ribosome IF1 helps IF2 and 3
Is it reasonable to expect that protein degradation can take place at any location in a cell?
If protein degradation took place at any location in a cell, indiscriminate breakdown of functional proteins could take place, so this is an unlikely occurrence. It is much more useful to the cell to have a mechanism for tagging proteins to be degraded and to do so at a specific location in the cell.
How is elongation different in prokaryotes and in eukaryotes
It is essentially the same - in eukaryotes there are corresponding EF's to carry out same functions
What is unique about selenocysteine?
It is unique chemically because it has a selenium ion in it, which takes the place of a sulfur in cysteine. It is also unique in that it appears to be coded for in the DNA sequence, even though the codon would normally be a stop codon.
Consider protein degradation in the absence of ubiquitinylation. Is the process likely to be more or less efficient?
Less. If proteins to be degraded did not have some signal marking them, the process would take place more randomly
Outline the proofreading processes in amino acid activation.
Proofreading in amino acid activation takes place in two stages. The first requires a hydrolytic site on the aminoacyl-tRNA synthetase; incorrect amino acids that have become esterified to the tRNA are removed here. The second stage of proofreading requires the recognition site on the aminoacyl-tRNA synthetase for the tRNA itself. The incorrect tRNA does not bind tightly to the enzyme.
E. coli has two tRNAs for methionine. What is the basis for the distinction between the two?
Methionine bound to tRNAfmet can be formylated, but methionine bonded to tRNAmet cannot be.
Benefit of having a wobble
Minimizes damage caused by misreading code -- ex: if Leu codon (CUU) misread as CUC, CUG, or CUA, still translated as Leu
Can the same enzyme esterify more than one amino acid to its corresponding tRNA?
No, A separate synthetase exists for each amino acid, and this synthetase functions for all of the different tRNA molecules for that amino acid.
3 binding sites for tRNA present on 50S subunit
P (peptidyl) site - the binding site on a ribosome for the tRNA that carries the growing peptide chain A (aminoacyl) site - the binding site for the aminoacyl-tRNA to be added to the growing peptide chain E (exit) site - the binding site for an uncharged tRNA about to be released from the ribosome
Identify the following by describing their functions: EF-G, EF-Tu, EF-Ts, EF-P, and peptidyl transferase.
Peptidyl transferase catalyzes the formation of a new peptide bond in protein synthesis. The elongation factors, EF-Tu and EF-Ts, are required for binding of aminoacyl tRNA to the A site. The third elongation factor, EF-G, is needed for the translocation step in which the mRNA moves with respect to the ribosome, exposing the codon for the next amino acid. EF-P is thought to help catalyze the formation of the first peptide bond.
Suggest a reason why the proofreading step in protein synthesis takes place at the level of amino acid activation rather than that of codon-anticodon recognition.
Proofreading at the activation step allows for selection of both the amino acid and the tRNA. If proofreading took place at the level of codon-anticodon recognition, there would not be a mechanism to ensure that the correct amino acid has been esterified to the tRNA
Protein synthesis takes place much more slowly in eukaryotes than in prokaryotes. Suggest a reason why this is so.
Protein synthesis in prokaryotes takes place as a coupled process with simultaneous transcription of mRNA and translation of the message in protein synthesis. In eukaryotes, mRNA is transcribed and processed in the nucleus and only then exported to the cytoplasm to direct protein synthesis.
Shine-Dalgarno sequence
Purine-rich leader sequence that precedes start signal (5'-GGAGGU-3') 10 nucleotides upstream of AUG start Acts as ribosomal binding site
Process of chain termination
RF-1 and 2 bind stop codons RF-3 bound to GTP facilitates activity of RF-1 and 2 Release factors hydrolyze peptide bond - Requires Gly-Gly-Gln - Peptide chain released from tRNA RRF - ribosome recycling factor - Facilitates complex dissociation thru GTP hydrolysis Releases RFs, tRNA, mRNA, 30S and 50S ribosomal subunits
What are two major similarities between protein synthesis in bacteria and in eukaryotes? What are two major differences?
Similarities: same start and stop codons; same genetic code; same chemical mechanisms of synthesis; interchangeable tRNAs. Major differences: in prokaryotes, the Shine-Dalgarno sequence and no introns; in eukaryotes, the -cap and -tail on mRNA and introns have been spliced out.
Why is it advantageous to have a mechanism to override the effect of stop codons in protein synthesis?
Some mutations can introduce stop codons. It is useful to a cell to have some mechanism to suppress the formation of incomplete proteins.
Wobble
Some tRNAs bond to one codon exclusively, but many of them can recognize more than one codon because of variations in the allowed pattern of hydrogen bonding. This variation is called wobble. - Applies to first base of anticodon (5' end) - pairs with third base in codon (3' end) Third base in codon can vary yet specify the same amino acid First base in anticodon of tRNA base pairs with wobble base
Chain initiation in prokaryotes
Synthesis starts at N-terminal end - initial N-terminal amino acid is always N-formylmethionine (fmet) Charged tRNAs devoted: fmet-tRNAfmet, it is fmet-tRNAmet if it is not first methionine Methionine bound to tRNAmet 1st, then formylated
What are the A site and the P site? How are their roles in protein synthesis similar? How do they differ? What is the E site?
The A site and the P site on the ribosome are both binding sites for charged tRNAs taking part in protein synthesis. The P (peptidyl) site binds a tRNA to which the growing polypeptide chain is bonded. The A (aminoacyl) site binds to an aminoacyl tRNA. The amino acid moiety is the next one added to the nascent protein. The E (exit) site binds the uncharged tRNA until it is released from the ribosome.
Why a degenerate genetic code helps protect an organism from the effects of mutations.
The degeneracy of the code acts as a buffer against deleterious mutations.
What is the role of ATP in amino acid activation?
The hydrolysis of ATP to AMP and PPi provides the energy to drive the activation step.
What are the components of the initiation complex in protein synthesis? How do they interact with one another?
The initiation complex in E. coli requires mRNA, the 30S ribosomal subunit, , GTP, and three proteininitiation factors, called IF-1, IF-2, and IF-3. The IF-3 protein is needed for the binding of mRNA to the ribosomal subunit. The other two protein factors are required for the binding of to the mRNA-30S complex
In the early days of research on protein synthesis, some scientists observed that their most highly purified ribosome preparations, containing almost exclusively single ribosomes, were less active than preparations that were less highly purified. Suggest an explanation for this observation.
The less highly purified ribosome preparations contained polysomes, which are more active in protein synthesis than single ribosomes.
Describe the recognition process by which the tRNA for N-formylmethionine interacts with the portion of mRNA that specifies the start of transcription.
The methionine anticodon (UAC) on the tRNA base pairs with the methionine codon AUG in the mRNA sequence that signals the start of protein synthesis.
Why do amino acids other than methionine occur in the N-terminal position of proteins from eukaryotes?
The original N-terminal methionine can be removed by posttranslational modification.
Why is selenocysteine called the 21st amino acid when there are many more amino acids found than the basic ones coded for by the genetic code?
The other amino acids found in proteins are created by modifying one of the twenty standard amino acids after the protein is made. Selenocysteine is formed while the amino acid is bound to tRNA. Thus, this amino acid is inserted into the protein during translation just like the standard 20 amino acids are.
Is amino acid activation energetically favored? Why or why not?
The overall process of amino acid activation is energetically favored because of the energy contributed by the hydrolysis of two phosphate bonds. Without that input of energy, it would not be favorable.
Describe the role of stop codons in the termination of protein synthesis.
The stop codons are recognized by proteins called release factors. A release factor not only blocks the binding of a new aminoacyl-tRNA but also affects the activity of the peptidyl transferase, so that the bond between the carboxyl end of the peptide and the tRNA is hydrolyzed.
Describe the role of the stop signals in protein synthesis.
The stop codons bind to release factors, proteins that block binding of aminoacyl tRNAs to the ribosome, and to release the newly formed protein
Coupled Transcription and Translation in Prokaryotes
This is possible in prokaryotes because of the lack of cell compartmentalization. In eukaryotes, mRNA is produced in the nucleus, and most protein synthesis takes place in the cytosol.
Is it reasonable that codons for the same amino acid have one or two nucleotides in common? Why or why not?
Yes it is reasonable. When codons for a given amino acid have one or two nucleotides in common, a mutation is less likely to give rise to a nonfunctional protein. The survival value of such a feature guarantees its selection in evolution.
Features of the genetic code
Triplet codon - sequence of 3 bases needed to specify 1 amino acid. Genetic code must translate language of 4-base DNA into language of 20-amino acid proteins. Degenerate - 64 possible triplets of four bases in RNA (4^3). 61 codons code for 20 amino acids. 3 stop codons - UGA, UAG, UAA. More than one triplet can encode same amino acid. However, each codon can specify only one amino acid. Nonoverlapping - No bases shared between consecutive codons. Ribosomes moves alone mRNA 3 bases at a time Comma-less - no intervening bases between codes Universality of code - code the same in all organisms, observed in viruses, prokaryotes, eukaryotes. except some codons in mitochondria different from those in nucleus
What is the value of the two stages in activation in terms of this accuracy?
Two stage amino acid activation reaction allows selectivity at two levels. Amino acid level -Aminoacyl-AMP remains bound to enzyme -Binding of correct amino acid verified by editing site in synthetase
What role does ubiquitin play in the degradation of proteins?
Ubiquitin is a small polypeptide (76 amino acids) that is highly conserved in eukaryotes. When ubiquitin is linked to a protein, it marks that protein for degradation in a proteasome.
Benefits of the 5' cap and 3' PolyA tail in eukaryotic mRNA
Untranslated regions ranging between 40 and 150 bases in length occur at both the 5' and 3' ends of the mature mRNA. An initiation codon at the 5' end, invariably AUG, signals the translation start site.
You are studying with a friend who says that the hydrogen-bonded portions of tRNA play no important role in its function. What is your reply?
Your friend is mistaken. The hydrogen-bonded regions contribute to the overall shape of the tRNA. Hydrogen-bonded regions are also important in the recognition of tRNAs by aminoacyl-tRNA synthetases
function of suppressor tRNA
allows translation to continue through a stop codon. tend to be found in cells in which a mutation has introduced a stop codon.
What does Shine-dalgarno sequence bind to
binds to pyrimidine-rich sequence on 16S rRNA of 30S subunit - aligns it for translation beginning with AUG codon
Wobble hypothesis
degenerate codons for given amino acid differ only in 3rd base - fewer unique tRNAs needed - tRNA can base-pair with several codons
initiator tRNA in prokaryotes
fMet-tRNAfmet (first aminoacyl-tRNA)
Nature of some of the modifications
fmet hydrolyzed in prokaryotes peptide bonds cleaved to produce truncated form leader sequences removed disulfide bonds formed prosthetic groups added amino acids modified carbohydrates or lipids added groups added (methyl, phosphoryl, etc.)
Selenocysteine
incorporated into protein S replaced by Se Does not have its own tRNA synthetase Made while bound to tRNA UGA codon - read differently due to special EFs
Two main pathways for protein degradation
lysosome and proteasome
Does mRNA bind to one or to both ribosomal subunits in the course of protein synthesis?
mRNA binds to the smaller ribosomal subunit.
Translocation
motion of the ribosome along mRNA as message read Occurs before next amino acid added performed by EF-G by GTP hydrolysis 1. Uncharged tRNA moves from P to E site, then released 2. Peptidyl-tRNA moves to vacant P site from A site
How many release factors are there in eukaryotes?
one
Transpeptidation
peptide bond formed in reaction catalyzed by peptidyl transferase alpha-amino group in A site performs nucleophilic attack on carbonyl in P site May be helped by rRNA of ribosome
Lysosome pathway
proteins directed to lysosome by signal sequence nonspecific once inside
proteasome pathway
proteins ubiquinylated (bind the polypeptide ubiquitin to a protein) to mark for degradation - involves multiple enzymes
How chaperones relate to degradation process
recognize unfolded proteins and help them fold and recognize misfolded proteins and send them to ubiquintinylation system
Synthetases have specific binding sites for ___
tRNA
The met attached to _____ first then formylated
tRNAfmet
Initiation complex
the aggregate of mRNA, N-formylmethione-tRNA, ribosomal subunits, and initiation factors needed at the start of protein synthesis
Polyribosomes/Polysome
the assemblage of several ribosomes bound to one mRNA
The fidelity of protein synthesis is assured twice during protein synthesis. How and when?
the first time during activation of the amino acids and the second time during the matching of the codon to the anticodon on the mRNA.