Lecture 23

Question 1

Nucleic vs amino acids

Answer 1

Nucleic acids have a lot of structural similarities between each base as they perform the same function. Side chains on a.a have wide diversity as they take place in a variety of processes.

Question 2

Codons

Answer 2

Needs min 3. nucleotides. If there were 2 positions instead of 3, you wouldn’t have enough permutations to code for 20 a.a (44 =16) therefore you need 3 (44*4 = 64). They also do not overlap i.e every set of 3 is read one codon and there’s no punctuation in genetic code i.e theres no specific nucleotide that is a stop signal and there’s directionality from 5’-3’. It is degenerate (3 nucleotides = 64 codons but only 20 a.a therefore most a.a encoded by >1 a.a), advantageous as if mutation occurs it decreases risk of producing truncated, dysfunctional protein.

Question 3

Error rate of translation

Answer 3

More complex therefore increases risk of error. Need to balance translation accuracy with speed (40 a.a/sec in E.coli). Translation error in E.Coli, must not excees 1 per 10000 a.a. Transcription error rate 1 in 10000 to 1 in 100000 bases.

Question 4

tRNAs

Answer 4

tRNA binds to specific codon and brings a.a with it on 3’ end. At least one tRNA for each a.a with anticodon in a central loop. They share very similar structural features as they have to interact the same way with mRNA the same way as with the ribosomes and the different protein factors that regulate the translation process. Similar structure incl. single-stranded molecule, ~73-93 ribonucleotides long, bases are modified and base pair with each other to form L-shaped receptor.

Question 5

Codon recognition by tRNAs and degeneracy

Answer 5

Codon and anticodon line up in antiparallel manner relative to codon. But, there are fewer tRNA compared to no. of codons therefore, some tRNAs must be able to recognise more than 1 codon and it doesn’t adhere to simple base pairing. Cells take advantage of degeneracy to decrease amount of tRNA molecules made. Recognition of 3rd base is less discriminating than first 2 positions.

Question 6

Wobble effect

Answer 6

There is more steric freedom and less stringency in that binding at the 3rd position of codon (1st anticodon position)

Question 7

Codon and tRNA interaction

Answer 7

Depending on 1st anticodon base, it dictates how many codons that tRNA can bind to. If first anticodon base is C, it will only bind to G and so on. I is degradation product of A and does not discriminate.

Question 8

Amino acid binding to tRNA

Answer 8

Binding of a.a to a particular tRNA establishes the genetic code. The a.a is attached via its carboxyl group to the 2’ or 3’ OH of the adenine at the CCA arm of the tRNA, forming an aminoacyl-tRNA ester. This reaction is catalysed by aminoacyl-tRNA synthetase and requires 2 ATP. Free a.a peptide bond formation is unfavourable without this activation step.

Question 9

A.a recognition by aminoacyl-tRNA synthetase

Answer 9

aminoacyl-tRNA synthetase must put correct a.a onto tRNA. Each enzyme is highly specific for a given a.a and uses specific a.a properties to connect it to the corresponding tRNA. Some a.a have same functional groups therefore, tRNA must be able to differentiate between them.

Question 10

Threonyl-tRNA synthetase

Answer 10

Distinguishes between threonine through Zn in activation site. Zn binds to amine and OH group on substrate. The OH group forms H bonds with a separate a.a that is also in active site of enzyme. Zn/Asp can differentiate Thr and Val but not Thr and Ser. AA-tRNA synthetase has proofreading function. If Ser is misincorporated, the CCA arm can swing from activation to editing site where Ser is cleaved. CCA can swing back and reattempt getting the correct a.a. Extra methyl makes Thr too big to fit into editing site therefore, cannot be cleaved off. Only a.a smaller than correct one will go to editing site, those that are too big won’t have functional groups that allow it to bind to Zn.

Question 11

tRNA recognition by aminoacyl-tRNA synthetase

Answer 11

Aa-tRNA synthetase recognise variable sites on tRNA and can determine which tRNA is appropriate. Increase changes = increase frequency it is used as recognition site.

Question 12

Ribosomes

Answer 12

Composed of RNA and protein. Coordinate interaction between mRNA and tRNA. Smaller (30S) + larger subunit (50S) is ribosome (70S). Key catalytic sites mainly composed of RNA with minor contributions from proteins.

Question 13

Bacterial translation

Answer 13

Pro translation and transcription occur simultaneously in the same space. Multiple ribosomes can translate a single mRNA strand. The 5’ end of mRNA interacts with ribosomes before transcription of the 3’ end is finished. Possible as both processes occur 5’-3’.

Question 13

Ribosomes bind tRNA and mRNA

Answer 13

They have 3 tRNA binding sites: A, P and E which span both subunits. mRNA bound in 30S subunit. tRNA in the A and P sites are bound to mRNA via anticodon-codon pairing.

Question 14

Initiation

Answer 14

Ribosomal complex assembles around mRNA and looks for start signal incl start codon which is ~25nt away from 5’ end.

Question 15

Elongation

Answer 15

mRNA is read 5’-3’ one codon at a time

Question 16

Termination

Answer 16

Stop codon reached and protein factors cleave the p.p from last tRNA, dissociates the pp and ribosomal complex from mRNA for another round of translation.

Question 17

Initiator codon

Answer 17

Usually AUG (codes for methionine). Binds to anticodon of first tRNA.

Question 18

Shine-Dalgarno Sequence

Answer 18

Purine (A/G) rich sequence centred ~10nt upstream of start codon. Pairs with 16s rRNA found in small ribosomal subunit.