Ch. 39 Textbook Problems
Codon
Three nucleotides
Translation
protein synthesis
A transfer RNA with a UGU anticodon is enzymatically conjugated to 14C-labeled cysteine. The cysteine unit is then chemically modified to alanine. The altered aminoacyl-tRNA is added to a protein-synthesizing system containing normal components except for this tRNA. The mRNA added to this mixture contains the following sequence: 5'-UUUUGCCAUGUUUGUGCU-3' What is the sequence of the corresponding radiolabeled peptide?
+H3N-Phe-Cys-His-Val-Ala-Ala-COO-
In a non overlapping triplet code, each group of three bases in a sequence ABCDEF... specifies only one amino acid - ABC specifies the first, DEF the second, and so forth - whereas, in a completely overlapping triplet code, ABC specifies the first amino acid, BCD the second, CDE the third, and so forth. Assume that you can mutate an individual nucleotide of a codon and detect the mutation in the amino acid sequence. Design an experiment that would establish whether the genetic code is overlapping or non overlapping.
- In a non-overlapping triplet genetic code, a sequence such as ABCDEF would code for two amino acids, one coded by ABC and the other by DEF - If we were to generate a mutation in the DNA sequence such that a different amino acid is coded for by the new codon, the amino acid sequence would be differentially affected in an overlapping or a non-overlapping code. - Thus, if the nucleotide C is mutated to X, in an overlapping genetic code the amino acids affected would be those coded by codons ABX, BXD, and XDE. That is three amino acids would be changed out of four. - In case of a non-overlapping code however, only ABX would be affected since the codon containing nucleotide C/X is coding for only one amino acid. This is thus a less deleterious approach and thus the non-overlapping code is prevalent in nature.
The code word GGG cannot be deciphered in the same way as can UUU, CCC, and AAA, because poly(G) does not act as a template for protein synthesis. Poly(G) forms a triple-stranded helical structure. Why is it an ineffective template?
- Only single stranded mRNAs can serve as templates for protein synthesis - Since poly G forms a triple stranded helix, it cannot serve as a template for protein synthesis
Using threonyl-tRNA synthetase as an example, account for the specificity of threonyl-tRNA formation.
- amino acids larger than the correct amino acid cannot fit into the active site of the tRNA - smaller but incorrect amino acids that may become attached to the tRNA fit into the editing site & are cleaved from the tRNA - e.g. serine may become attached to the threonyl-tRNA, but the serine will fit into the editing site & be cleaved from the threonyl-tRNA - when the correct threonine is attached, it will not fit the editing site & will not be cleaved
What are key characteristics of the genetic code?
1. an amino acid is encoded by three nucleotides 2. the code is non overlapping 3. there is no punctuation in the code 4. there is directionality in the code (5' to 3') 5. the genetic codes are degenerates in nature
Aminoacyl-tRNA synthetases are the only components of gene expression that know the genetic code. Explain.
- aminoacyl-tRNA synthetases are the only components that actually match a nucleotide sequence (the three-base RNA anticodon) with a particular amino acid to define the genetic code - all other interactions of genetic code components involve simply "Watson-Crick" pairing between complementary bases
Synthetic rna molecules of defined sequence were instrumental in deciphering the genetic code. Their synthesis first required the synthesis of DNA molecules to serve as a template. H. Gobind Khorana synthesized, by organic-chemical methods, two complementary deoxyribonucleotides, each with nine residues: d(TAC)3 and d(GTA)3. Partly overlapping duplexes that formed on mixing these oligonucleotides then served as templates for the synthesis by DNA polymerase of long, repeating double-helical DNA strands. The next step was to obtain long polyribonucleotide strands with a sequence complementary to only now of the two dna strands. How did Khorana obtain only poly(UAC)? Only poly(GUA)?
- note that each complementary strand is missing 1 of the 4 bases: d(TAC) lacks G and d(GTA) lacks C - so incubation with RNA polymerase and only UTP, ATP, and CTP lead to the synthesis of only poly UAC, the RNA complement of d(GTA) - when GTP was used in place of CTP, the complement of d(TAC), poly(GUA), was formed
RNA is readily hydrolyzed by alkali, whereas DNA is not. Why?
The 2'-OH in RNA acts as an intramolecular nucleophile In the the alkaline hydrolysis of RNA, the 2'-OH groups forms a 2'-3' cyclic intermediate
What features are common to all tRNA molecules?
1. all of the tRNA molecules consist of a single RNA chain that contains 73 to 93 ribonucleotides 2. the three dimensional structure of tRNAs is L-shaped; while in two dimensions they possess a clover leaf structure 3. the presence of unusual bases is a unique property of tRNA molecules. They contain A, U, G, and C bases modified by methylation, a modification that facilitates interactions with other components of the translation machinery. Other modified bases such as inosine are also present. 4. the clover lead structure of tRNA molecules consists of double stranded helix portions and 5 unpaired regions: the 3' CCA terminal region, the TWC? loop, the extra arm, the DHU loop, and the anticodon loop 5. the tRNA usually has a phosphorylated 5' end with the terminal residue usually being guanosine 6. the 3' OH of the CCA end of the tRNA is attached to the activated amino acid forming the aminoacyl tRNA molecule
The RNA transcript of a region of T4 phage dna contains the sequence 5'-AAAUGAGGA-3'. In theory, this sequence is capable of encoding three different polypeptides. What are they?
1. the peptide chain is terminated with AAA that is Lys. It also has a UGA stop codon. 2. Here AAU encoded for Asn and GAG encoded for Glu, so the polypeptide is Ans-Glu 3. Here AUG encoded for Met and AGG encoded for Arg, so here the polypeptide is Met-Arg.
Calculate the probability of synthesizing an error-free protein of 50 amino acids and one of 300 amino acids when the frequency of inserting an incorrect amino acid is 10^-2. Repeat the calculations with error frequencies of 10^-4 and 10^-6.
? figure out
Why has the code remained nearly invariant through billions of years of evolution, from bacteria to human beings?
A mutation that altered the reading of mRNA would change the amino acid sequence of most, if not all, proteins synthesized by that particular organism. Many of these changes would undoubtably be deleterious, and so there would be strong selection again a mutation with such pervasive consequences. It allows the genetic code to instead use its energy for things like dna replication and species preservation.
Explain how it is possible that some tRNA molecules recognize more than one codon.
Because of wobbles. When there is freedom in the third position of codon, it is referred to as wobble. For example, yeast alanyl-tRNA has anticodon IGC, here I is inosine nucleoside. And this anticodon binds with the three probable codons - GCU, GCC, and GCA. The first two bases in a codon form are checked for fidelity by bases of the 16s rRNA. The third base is not inspected for accuracy, so some variation is tolerated.
tRNA
Carrier of activated amino acids
30S subunit
Contains 16S RNA
50S subunit
Contains 28S and 5S RNA
Usually degeneracy should be avoided, except perhaps in well-controlled circumstances. However, it is advantageous for the genetic code to be degenerate. What does it mean to say that the code is degenerate, and explain why a degenerate code is valuable?
Degeneracy of the code means that for most amino acids, there is more than one codon. This property is valuable because if the code were not degenerate, 20 codons would encode amino acids and the rest of the codons would lead to chain termination. Most mutations would then likely lead to inactive proteins.
What two reaction steps are required for the formation of an aminoacyl-tRNA?
First is the formation of the aminoacyl adenylate, which then reacts with the tRNA to form the aminoacyl-tRNA Both steps are catalyzed by aminoacyl-tRNA synthetase Amino acid + ATP + tRNA aminoacyl-tRNA + AMP + PPi
The energetic equivalent of two molecules of ATP is used to activate an amino acid, yet only one molecule of ATP is used. Explain.
In the process of the activation of amino acids, it is catalyzed by aminoacyl-tRNA synthetase. In the first step from amino acid and ATP aminoacyl adenylate forms tat is also known as aminoacyl-AMP. So Amino acids + ATP <---> aminoacyl-AMP + PPi So in the process of the activation of amino acids, only one molecule of ATP is used. But we know that two molecule of ATP are used to activate an amino acid. The second ATP is used to convert AMP into ADP, which is known as the substrate for oxidative phosphorylation.
Proteins generally have low contents of Met and Trp, intermediate ones of His and Cys, and high ones of Leu and Ser. What does this observation suggest about the relation between the number of codons of an amino acid and it frequency of occurrence in proteins? What might be the selective advantage of this relation?
Highly abundant amino acid residues have the most codons (eg Leu and Ser each have 6) whereas the least abundant amino acids have the fewest (Met and Trp each have only one) Degeneracy (1) allows variation in base composition (2) decreases the likelihood that a substitution for a base will change the encoded amino acid. If the degeneracy were equally distributed, each of the 20 amino acids would have 3 codons. Both benefits (1 and 2) are maximized by the assignment of more codons to prevalent amino acids than to less frequently used ones.
Why is it crucial that protein synthesis has an error frequency of 10^-4?
If there was a higher error rate, then there are too many defects in proteins. And the lower rates are possible only by the reduction in the rate of the synthesis of proteins. It is because the additional time is required to read the errors.
In the context of protein synthesis, what is meant by an activated amino acid?
In the process of protein synthesis, the amino acids are required to attach with specific tRNA molecules. The activation of amino acids is catalyzed by specific enzymes known as aminoacyl-tRNA syntheses. This linkage between amino acid and tRNA acts as a translation step or actually substrates in protein synthesis. Hence, in the process of protein synthesis, the activated amino acids are known as the amino acids that are linked with appropriate tRNA.
A series of experiments were performed to establish the direction of chain growth in protein synthesis. Reticulocytes (young red blood cells) that were actively synthesizing hemoglobin were treated with [3H]leucine. In a period of time shorter than that required to synthesize a complete chain, samples of hemoglobin were taken, separated into alpha and beta chains, and analyzed for the distribution of 3H within their sequences. In the earliest samples, only regions near the carboxyl ends contained radioactivity. In later samples, radioactivity was present closer to the amino acid terminus as well. Explain how these results determine the direction of chain growth in protein synthesis.
In the reticulocytes, the synthesis of hemoglobin is on-going even before the addition of labeled leucine. Thus, when 3H-leucine is added initially and determined for incorporation in incomplete hemoglobin chains, only the leucines occurring at the carboxyl terminal end of the chains show labelling, since carboxyl terminals are the last to be translated. As time passes though, the presence of label leucine in the cell allows its incorporation even into N-terminal amino acid sequences containing leucine. This is because the labelled amino acid is present at the time of translation initiation too. This proves that the incorporation of amino acids and chain growth occur in the amino acid terminal to carboxy terminal direction.
Genetic Code
Nucleic acid information to protein information
Look at question 26
The polynucleotide 5' A-A-A-(-A-A-A-) n-A-A-C 3' consists of AAA codon at the 5' end followed by poly - (-A-A-A-) - nucleotide chains. At the 3' end however, there is a C nucleotide making the last codon AAC. Since, AAA codes for lysine and AAC codes for asparagine, if the sequence is translated in the 5' to 3' direction, the polypeptide sequence would be: +H3N- Lys - (-Lys-) n - Asn - COO- With lysine at the amino end and throughout the protein and asparagine at the carboxyl end. Since the protein formed in the one above, it can be said that the translation of mRNA occurs in the 5' to 3' direction with the 5' end codon coding for the amino terminus amino acid and the 3' end codon before the stop codon coding for the carboxyl terminus amino acid.
What is the minimum number of contiguous bases required to encode 20 amino acids? Explain your answer.
There are 4 continuous bases. So the answer is 3 because 4^3 = 64. 4^2 doesn't work because that would only encode 16 amino acids.
Why must tRNA molecules have both unique structural features and common structural features?
Unique features are required so that the aminoacyl-tRNA synthetases can distinguish among the tRNAs and attach the correct amino acid to the correct tRNA. Common features are required bacause all tRNAs must interact with the same protein-synthesizing machinery.
Look at question 9
a. 5'- UAACGGUACGAU -3' b. Leu-Pro-Ser-Asp-Trp-Met c. Poly (Leu-Leu-Thr-Try)
Look at question 24
a. When ATP and 32PPi are incubated with the specific isoleucyl-tRNA synthetase enzyme, the label will not be incorporated in the ATP molecule. This is because the conversion of ATP to PPi is not a reversible reaction and thus ATP is not reformed from its components. The following reaction is not possible. Thus, labeled 32PPi will not be incorporated into the ATP molecule. b. In this case too, the label will not be incorporated into the ATP molecule. This is because the presence of tRNA does not facilitate the conversion of ATP to PPi and vice versa. Thus, there is no means of the dissociation and reformation of ATP into and from PPi. Therefore, labeled PPi cannot enter the ATP in this molecule either. c. In the presence of isoleucine, ATP, and PPi, the following reaction catalyzed by isoleucyl-tRNA synthetase will take place. Isoleucine + ATP Isoleucine-AMP + PPi Since the reaction is reversible, as shown in equations 1 and 2, the reverse reaction will also take place: Isoleucine-AMP + 32PPi Isoleucine + AT32P Thus, since the medium contains labeled 32PP, it will also be incorporated into the ATP molecule when the reverse reaction takes place.
Anticodon loop
interacts with the codon
aminoacyl-tRNA synthetase
code reader
Wobble
pairing freedom
Ribosome
site of protein synthesis
