LECTURE 14

Question 1

how many AAs are added per minute and second during translation?

Answer 1

15AA/second
900/minute

Question 2

how far apart are ribosomes on mRNA?

Answer 2

80nt apart

Question 3

in which direction are proteins sythesized?

Answer 3

from the N terminus to the C terminus

Question 4

what are the three steps of translation

Answer 4

initation elongation termination

Question 5

where does the ribosome bind to start translation?

Answer 5

before the AUG there is the shine dalgarno sequence in the leader region of the mRNA
the 16S rRNA of the small subunit recognises this sequence and that’s where it binds

Question 6

what is the length of tRNA?

Answer 6

between 73 and 93nt
forms a cloverleaf secondary structure (multiple stem loops)

Question 7

where are amino acids attached on the tRNA and how are they added there?

Answer 7

attached to the 3’ end
aminoacyl tRNA synthetase recognises the tRNA (recognises the anticodon) and loads it with the corresponding AA

Question 8

what is the structure of the E.coli ribosome?

Answer 8

total size: 70S
big subunit: 50S
5S rRNA (120nt)+23rRNA (2300nt)+34 polypeptide chain

small subunit: 30S
16SrRNA (1600nt)+21 polypeptide chain

the rRNAs act as scaffolds, and the polypeptides bind to them

(S=svedberg sedimentation factor)

Question 9

how does translation work?

Answer 9

1. at the P site is the tRNA with the growing polypeptide chain
2. a new tRNA binds to the A site
3. the chain from the E site is transferred to the new amino acid on the A site (by nucleophilic attack of the amino group of the new AA)
4. the tRNA on the E site is now free to leave
5. the ribosome moves 3 nucleotides forward and the growing chain is now on the E site, with the A site free for tRNA binding

Question 10

why are not all genes turned on?

Answer 10

that takes a lot of energy, the genes are only turned on when the protein is needed

Question 11

how does negative control by induction work and an example of that?

Answer 11

the gene is turned on when the enzyme is needed (substrate present/inducer)
- if the repressor is active it will bind the operator region of the gene and silence transcription
- if the inducer is present it will bind to repressor to inactivate it, which will not bind to the gene
the mRNA will then be synthesized and there will be translation of this gene

an example of that is lactose:
lactose is the inducer which will inactivate the repressor, leading to the transcription of that gene to make the corresponding mRNA

Question 12

how does negative control by repression work and an example of that?

Answer 12

the gene is turned on when the end product accumulates (corepressor)

• when the inactive repressor is bound to the corepressor, it will be activated, bind to the operator region and the gene will not be transcribed
• when the inactive repressor is not bound to anything, it is inactive and that gene will be transcribed

an example of that is tryptophan
tryptophan, when it accumulates too much, acts as a corepressor

Question 13

how does regulation of mRNA work by positive control and an example of that?

Answer 13

the gene is turned on only in the presence of a controlling factor (activator protein)
- when glucose is low there is high cAMP, which means that lac genes need to be activated to do lactose metabolism
- cAMP will bind to the CAP protein to activate it
- the active CAP will bind to the promoter region and recruit the RNA polymerase to start transcription

Question 14

how does regulation of mRNA work by attenuation?

Answer 14

initiation and continuation of transcription are controlled
more like a dimmer, more fine tuning than simply turning it on and off

Question 15

where does attenuation happen?

Answer 15

in the leader region of the mRNA

Question 16

how is expression of tryptophan controlled by attenuation?

Answer 16

1. the tryptophan operon is made of 5 genes, which are preceded by the region where negative control works and the trpL region, which is where attenuation happens
2. attenuation happens when there are high levels of tryptophan
3. in the leader sequence of the gene, the ribosomes read through that sequence and see how many tRNAs are bound to Trp
4. the leader region can have two forms
5. if there is a lot of Trp already, the ribosome will fall off and region 1 and 2 will complementary base pair together,which will create a Rho independent terminator factor later in the leader sequence, which leads to a stop in transcription
6. if there isn’t a lot of Trp, region 1 is transcribed, so regions 2 and 3 will pari together, which does not lead to a stop and the rest of the gene can be transcribed
7. these two forms are in equilibrium

Question 17

picture of attenuation process

Answer 17