BIOC 299 Chapter 39
genetic code
the relation between nucleic acid sequence information & protein sequence information
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
What is the minimum number of contiguous bases required to encode 20 amino acids? explain
- 3 contiguous bases - because there are 4 different bases, a code based on a 2-base codon could encode only 4^2 = 16 amino acids - a 3-base codon allows 4^3 = 64 combinations which is more than enough for the 20 amino acids
ribosome
- a large ribonucleoprotein assembly that catalyzes the formation of peptide bonds - a molecular machine that coordinates protein synthesis
Why has the code remained nearly invariant through billions of years of evolution, from bacteria to human beings?
- a mutation that altered the code for 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 undoubtedly be deleterious - the organisms would not survive & so there would be strong selection against a mutation with such pervasive consequences
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
aminoacyl-tRNA synthetase
- an enzyme that activates an amino acid that links into tRNA - also known as an activating enzyme, each aminoacyl-tRNA synthetase is specific for a particular amino acid
Why is it crucial that protein synthesis has an error frequency of 10^-4?
- an error frequency of 1 incorrect amino acid every 10^4 incorporations allows for the rapid & accurate synthesis of proteins as large as 1000 amino acids - higher error rates would result in too many defective proteins - lower error rate would likely slow down the rate of protein synthesis without a significant gain in accuracy
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, & explain why a degenerate code is valuable?
- degeneracy of the code means that, for many of the amino acids, there is more than 1 codon - the property is valuable because, if the code were not degenerate, only 20 codons would encode amino acids & the rest of the codons could lead to chain termination - many mutations would then lead to inactive proteins
What features are common to all tRNA molecules?
- each is a single chain of RNA - contain unusual bases - about half of the bases are base-paired to form double helices, the strands that comprise the double-helical segments are antiparallel to each other - the 5' end of tRNA is phosphorylated & is usually pG - the 3' end of the tRNA terminates with the sequence CCA, & the hydroxyl group of the A residue in the acceptor site for attachment of the incoming cognate amino acid - the anticodon is located in a loop near the center of the tRNA sequence
Proteins generally have low contents of Met & Trp, intermediate ones of His & Cys, & higher ones of Leu & Ser - what does this observation suggest about the relation between the number of codons of an amino acid & its frequency of occurrence in proteins? What might be the selective advantage of this relation?
- highly abundant amino acid residues have the most codons (e.g. Leu & Ser each have 6), whereas the least-abundant amino acids have the fewest (Met & Trp each have only 1) - degeneracy allows variation in base composition & 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 are maximized by the assignment of more codons to prevalent amino acids than to less frequently used ones
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, 2 complementary deoxyribonucleotides, each with nine residues: d(TAC)3 & d(GTA)3 Partly overlapping duplexes that formed on mixing these oligonucleotides then served as templates for the synthesis by DNA polymerase of long, repeated double-helical DNA strands The next step was to obtain long polynucleotide strands with a sequence complementary to only 1 of the 2 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 & only UTP, ATP & CTP led 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
The code word GGG cannot be deciphered in the same way as can UUU, CCC, & AAA, because poly G does not act as a template for protein synthesis, poly G forms a triple-stranded helical structure - why is it not 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
The RNA transcript of a region of T4 phage DNA contains the sequence 5'-AAAUGAGGA-3' In theory, this sequence is capable of encoding 3 different polypeptides - what are they?
- since 3 different polypeptides are synthesized, the synthesis must start from 3 different reading frames - one of these will be in phase with the AAA in the sequence shown in the problem & will therefore have a terminal lysine, since UGA is a stop signal - the reading frame in phase with AAU will result in a polypeptide having an Asn-Glu sequence in it, & the reading frame in phase with AUG will have a Met-Arg sequence in it
ribosomal RNA (rRNA)
- the RNA components of ribosomes - in eukaryotes, 18S RNA is a component of the 40S subunit, and 5S RNA, 28S RNA & 5.8S RNA are components of the 60S subunit - a site on the 28S RNA catalyzes peptide-bond formation
transfer RNA (tRNA)
- the adaptor molecule in protein synthesis - contains an amino-acid-recognition site as well as a template-recognition site or anticodon
Explain how it is possible that some tRNA molecules recognize more than 1 codon
- the first 2 bases in a codon form Watson-Crick base pairs that are checked for fidelity by bases of the 16S rRNA - the third base is not inspected for accuracy, & so some variation is tolerated - non-canonical base pairs are sometimes permitted in the third position
What 2 reaction steps are required for the formation of an aminoacyl-tRNA?
- the first step involves the formation of an aminoacyl adenylate, an intermediate that is formed by reaction between an amino acid & ATP, with release of pyrophosphate - the aminoacyl adenylate then reacts the tRNA to form the aminoacyl-tRNA - both steps are catalyzed by an aminoacyl-tRNA synthetase that is specific for a particular amino acid & its cognate tRNA molecules
50S subunit
- the large subunit of the bacterial 70S ribosome - the site of peptide bond synthesis, it contains 34 different proteins, a 5S RNA species, & a 23S RNA species
30S subunit
- the small subunit of the bacterial 70S ribosome - composed of 21 different proteins & a 16S RNA molecule
A transfer RNA with a UGU anticodon is enzymatically conjugated 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?
- the triplet codons 5'-UUU-UGC-CAU-GUU-UGU-GCU-3' would be translated in sequence as: Phe-Ala-His-Val-Ala-Ala - the codons UGC & UGU encode cysteine but, because the cysteine has been modified to alanine on the aminoacyl-tRNA, alanine is incorporated in place of cysteine at the underlined positions in the sequence
In a nonoverlapping triplet code, each group of 3 bases in a sequence ABCDEF... specifies only 1 amino acid - ABC specifies the first, DEF the second, etc. - whereas, in a completely overlapping triplet code, ABC specifies the first amino acid, BCD the second, CDE the third, etc. Assume that you can mutate an individual nucleotide of a codon & detect the mutation in the amino acid sequence - design an experiment that would establish whether the genetic code is overlapping or nonoverlapping
- these alternatives can be distinguished based on the results of studies of the sequences of amino acids in mutants - suppose that the base C in the given sequence is mutated to C' - in a nonoverlapping code, only amino acid 1 will be changed - in a completely overlapping code, amino acids 1, 2 & 3 will all be altered by a mutation of C to C' - the results of amino acid sequence studies of tobacco mosaic virus mutants & abnormal hemoglobins showed that alterations usually affected only a single amino acid - the genetic code was concluded to be nonoverlapping
Why must tRNA molecules have both unique structural features & common structural features?
- unique features are required so that the aminoacyl-tRNA synthetases can distinguish among the different tRNA molecules & attach the correct amino acid to the proper tRNA - common features are required because all of the aminoacyl-tRNAs then must interact with the same protein-synthesizing machinery in the ribosomes
The energetic equivalent of 2 molecules of ATP is used to activate an amino acid, yet only 1 molecule of ATP is used - explain
- when the single ATP molecule is cleaved to AMP & pyrophosphate, the pyrophosphate is subsequently hydrolyzed to give 2 molecules of orthophosphate - the effective cost is 2 high-energy phosphate bonds - a second ATP molecule is then required to convert the AMP into ADP, the substrate for oxidative phosphorylation
1. Write the sequence of an mRNA molecule synthesized from a DNA template strand having the following sequence: 5'-ATCGTACCGTTA-3' 2. What amino acid sequence is encoded by the following base sequence of an mRNA molecules? assume that the reading frame starts at the 5' end: 5'-UUGCCUAGUGAUUGGAUG-3' 3. What is the sequence of the polypeptide formed on addition of poly(UUAC) to a cell-free protein-synthesizing system that does not require a start codon?
1. because mRNA is synthesized antiparallel to the DNA template & A pairs with U & T pairs with A, the correct sequence is 5'-UAACGGUACGUA-3' 2. Since the 5' end of an mRNA molecule codes for the amino terminus, appropriate use of the genetic code leads to Leu-Pro-Ser-Asp-Trp-Met 3. Since one has a repeating tetramer (UUAC) & a 3-base code, repetition will be observed at a 12-base interval (3 x UUAC), comparison of this 12-base sequence with the genetic code leads to the conclusion that a polymer with a repeating tetrapeptide (Leu-Leu-Thr-Tyr) unit will be formed
What are the key characteristics of the genetic code?
3 consecutive nucleotides encode an amino acid, nonoverlapping, no punctuation, & degenerate
anticodon loop
3 nucleotide sequence of tRNA that base pairs with a codon in mRNA
polysome
a group of ribosomes bound to an mRNA molecule & simultaneously carrying out translation
codon
a nucleotide triplet in mRNA that encodes a particular amino acid
In the context of protein synthesis, what is meant by an activated amino acid?
an activated amino acid is one that is covalently coupled to its appropriate tRNA molecule
translation
cellular protein synthesis, so named because the 4 letter alphabet of nucleic acids is translated into the different amino acids that make up proteins
wobble
refers to steric freedom in the pairing of the third base of an mRNA codon with the anticodon of a tRNA molecule, which allows more than 1 codon to be recognized by a particular tRNA molecules
Calculate the probability of synthesizing an error-free protein of 50 amino acids & one of 500 amino acids when the frequency of inserting an incorrect amino acid is 10^-2 - repeat the calculations with error frequencies of 10^-4 & 10^-6
the probability is calculated with the equation p = (1 - ε)^n, where p is the probability of synthesizing the error-free protein, ε is the error rate, & n is the number of amino acid residues in the protein: 0.605, 0.0066, 0.995, 0.951, 0.999, 0.999