Translation (Dr. Ruddy)

Question 1

Is RNA transcribed form the template (non coding) strand or the coding strand ?

Answer 1

The template strand (which is thus complementary to RNA). The coding strand thus has the same sequences as the RNA, with Ts instead of Us.

Question 2

Which strand is sense and antisense ?

Answer 2

The coding strand is sense and the template strand is antisense.

Question 3

How many codons can be made with 4 nts ?

Answer 3

4ˆ3 = 63

Question 4

How many sop codons are there ?

What are they ?

Answer 4

3: UGA, UAA, UAG

Question 5

Why is the genetic code “degenerate” ?

Answer 5

Because several codons can be used to specify one amino acid (out of 64-3=61 possibilities, there are only 20 amino acids).

Question 6

How many reading framed are there ?

Answer 6

Since a codon is three nucleotides, there are three reading frames (e.g. abc abc abc or bca bca bca or cab cab cab).

Question 7

Which reading frame is correct ?

Answer 7

Most RNAs only give one possible reading frame, because stop codons in the two other possible reading frames would terminates translation before a functional proteins is produced. However, this does happen in bacteria, that have a much smaller genome and where cell division is a rate limiting event.

Question 8

What types of DNA mutations can occur during translation ?

Answer 8

Frameshift mutations (by addition of deletion of a nt), missense mutations (which can be silent because of the degeneracy of the genetic code) and nonsense (insertion of a stop codon in the middle of a sequence).

Question 9

Which proteins are absolutely vital to convert an mRNA sequence into a protein ?

Answer 9

rRNA, tRNA, and aminoacyl-tRNA acetyl synthetases (these load each tRNA with its appropriate amino acid).

Question 10

What are ribosomes and what is their function ?

Answer 10

Ribosomes are ribonucleoproteins made of 2 subunits, several different RNAs and more than 50 proteins. Their job is to synthesize proteins, and they do this at a rate of 3-5 AA/second.

Question 11

What is the difference between eukaryotic and prokaryotic ribosomes ?

Answer 11

Eukaryotic ribosomes are larger. They consist of a 60S large subunit and a 40S small subunit, which come together to form an 80S particle having a mass of 4200 kd, compared with 2700 kd for the prokaryotic 70S (50S+30S) ribosome. The 40S subunit contains an 18S RNA that is homologous to the prokaryotic 16S RNA.

Question 12

What two functions do all tRNAs have ?

Answer 12

Firstly, to covalently link (with a high energy ester bond) with an AA (brought by aminoacyl-tRNA synthases). This linkage requires hydrolysis of ATP to AMP. Secondly, to base pair with a codon in the mRNA.

Question 13

How specific are aminoacyl-tRNA synthetase ?

Answer 13

Each of the 20 aminoacyl-tRNA synthetases recognizes one amino acid and all of the compatible (cognate) tRNAs.

Question 14

What is “wobble” ?

Answer 14

The first two positions of the mRNA codon observe Watson-Crick base pairing rules (A-U, C-G) It is usually the third base of the codon (or first of the anti-codon) that exhibits wobble. Wobble occurs because the conformation of the tRNA anticodon loop permits flexibility at the first base of the anticodon. Wobble essentially happens because a single tRNA can recognize more that one codon corresponding to a different AA.

Question 15

Do all cells contain 61 tRNAs ?

Answer 15

No, some cells contain fewer than 61 tRNAs, and that is where the “Wobbles Hypothesis” comes into play.

Question 16

What are exemples of wobble pairing ?

Answer 16

Inosine (deaminated adenosine) paring with C, A and U. Uridine (nucleoside) pairing with G. Of course these pairing are not as good (hence wobble) but some interaction still happens and codon/anticodon binding is still possible.

Question 17

How do protein chains start in almost all cells ?

Answer 17

By the start codon, AUG, which codes for Methionine (Met or M), or f-Met in prokaryotes (N-Formylmethionine) in prokaryotes.

Question 18

What are the four steps of translation ?

Answer 18

Initiation, elongation, termination and recycling of translational components.

Question 19

How does the ribosome know which Met is the initiator AA ?

Does this mechanism differ between eukaryotes and eukaryotes ?

Answer 19

By recognition of specific sequences.
In bacteria, the 16S rRNA recognizes and base-pairs with the Shine-Dalgarno sequence (5’-AGGAGGU-3’) in the mRNA near the AUG initiator.
In eukaryotes, the ribosome bind to the mRNA near the 5’ capped end and translation starts at the Kozak sequence (after Marilyn Kozak) : 5’-GC(G or A)CCCAUGG- 3’.

Question 20

What are the 4 main sites of the ribosome ?

Answer 20

The A (aminoacyl) site, the P (peptidyl) site, the E (exit) site and the mRNA binding site.

Question 21

What kind of inhibitors can act of the ribosome ?

Answer 21

Antibiotics (erythromycin), toxins (Ricin, diphteria) and viruses (Hepatitis C)

Question 22

How does the ribosome recognize stop codons ?

Do eukaryotes and prokaryotes hav different recognition mechanisms ?

Answer 22

By specific termination release factors that promotee cleavage of the peptidyl-tRNA.
In prokaryotes, RF1 recognizes UAG/UAA, RF2 recognizes UGA/UAA, and RF3 is a GTP-binding protein that leads to the dissociation of RF1/RF2 after peptide release.
In eukaryotes, eRF1 recognizes all three termination codons and eRF3 is a ribosome-dependent GTPase that helps eRF1 release the completed polypeptide.

Question 23

What is western blotting ?

Answer 23

Western blotting is a technique used to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.
The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present.

Question 24

What is proteomics ?

Answer 24

Proteomics is a field of molecular biology concerned with the systematic, high-throughput approach to protein expression analysis of a cell or an organism.