Molecular Genetics (Andy Bailey 13-15)

Question 1

How is tryptophan (Trp) biosynthesis regulated in E.coli by feedback inhibition?

Answer 1

-Feedback inhibition:
accumulation of Trp slows down the rate of catalysis of the first enzyme complex (Trp D/E), reducing the rate of production.

Question 2

How is tryptophan (Trp) biosynthesis regulated in E.coli by transcriptional inhibition?

Answer 2

• TrpR (Trp regulator) is always switched on.
• Trp binds to a repressor (produced by the TrpR) and activates it
• Repressor binds to the operon and blocks translation.
• No tryptophan = operon expressed = polypeptides produced which are involved with the synthesis of tryptophan.

Question 3

Glucose is the preferred carbon source for E.coli, but lactose is also used (see lectures 1-4). How are glucose levels monitored?

Answer 3

The Lac operon monitors glucose by cyclic AMP (cAMP).
As glucose falls, cAMP increases, which helps switch on alternative pathways. Active CRP (cyclic AMP receptor protein) helps RNA polymerase bind to the Lac promoter.

Question 4

What are sigma factors?

Answer 4

They are bacterial transcription initiators, that enable specific binding of RNA polymerases to promoters.

E.coli have many sigma factors which can be used to coordinate gene families.
(rpoH- for heat-shock genes; rpoF- flagella genes for motility etc)

Question 5

What is Quorum Sensing?

Answer 5

A form of bacterial communication.
Cell density (of bacteria) can control gene expression in this way; they can hide until the colony is large enough to 'attack'.
Signal proteins ( e.g. AHL) are produced at low levels. the receptor proteins are only activated when AHL accumulates above a threshold level.

Question 6

In eukaryotes, promoters can be large and hard to define as they may include regions upstream and downstream of the gene. How can regions downstream be part of the promotor?

Answer 6

DNA-bending proteins may fold the DNA so that enhancers downstream of the gene are close to the promotor and help recruit polymerase.

Question 7

In eukaryotes, how do the 5’ and 3’ modifications help the stability of RNA?

Answer 7

enzymes recognise the 3’ UTR (Untranslated region), cleaves the end and replaces it with a polyA tail.
If the 5’ cap or polyA tail are absent, mRNA is likely to be digested.
This reduces the amount of faulty mRNA.

Question 8

Which codon is usually associated with translation initiation?

Answer 8

ATG

Question 9

What is the role Ubiquitin?

Answer 9

It mediates protein degradation in cells.

Activated ubiquitin is transferred to a conjugating enzyme, this then interacts with ubiquitin ligase. The ligase delivers the activated ubiquitin to the target substrate (the unwanted protein). this occurs several times, building a chain of ubiquitin, tagging the protein.
The protease system degrades proteins tagged by ubiquitin and the ubiquitin is recycled.

Question 10

Why may proteins be targeted for degradation by ubiquitin?

Answer 10

• If there are too many
• if they’re folded in the wrong way/wrong shape
• if they’re degraded.

Question 11

How does acetylation of chromatin affect gene expression?

Answer 11

acetyl groups are attached to an amino acid in the histone tails. This opens up chromatin, promoting transcription by making it accessible to RNA polymerase.

Question 12

How does methylation of chromatin affect gene expression?

Answer 12

it condenses chromatin so the genes can’t be expressed

Question 13

Which 4 enzymes directly regulate chromatin structure?

Answer 13

HATs: histone acetyltransferases
HDACs: histone deacetylases
HMTs: histone methyltransferases
HDMCs: histone demethylases

Condenses chromatin: HDACs & HMTs
Opens chromatin: HATs & HDMCs

Question 14

Name 2 chemical modifiers and 2 genetic modifications for chromatin structure.

Answer 14

Chemical modifiers:

• SAHA; inhibits HDACs so chromatin stays acetylated for longer.
• SAC inhibits HMTs so chromatin is not so condensed.

Genetic (Both reduce fitness):

• Disruption of HDAC to maintain expression.
• Disruption of LacA to reduce methylation.

Question 15

Methylation has a regulatory ability in plants and animals. Methylation patterns are heritable, but if patterns are wrong what can it cause?

Answer 15

Cancers, infertility etc

It silences genes.

Question 16

Why is northern blotting used?

Answer 16

To analyse RNA. It reveals size and abundance of transcripts, but can only do on gene at a time. We can hybridise probes onto the RNA and an X-ray can be used.

Question 17

How can oligo(dT) be used to purify mRNA?

Answer 17

Oligo(dT) is a primer which catches the polyA tails of mRNA molecules. The polyA tails are only found on mRNA and act as a hook. This allows purification from tRNA and rRNA etc.

Question 18

Which process is used to produce DNA from mRNA?

Answer 18

Reverse transcription via reverse transcriptase. Can produce a copy of dsDNA.

Question 19

How can we use microarrays to measure the abundance of transcript for every gene?

Answer 19

Microarrays:

DNA from every gene is spotted onto a solid support. mRNA is labelled. Chips hybridise to the labelled mRNA and the signal from each spot is measured.

Question 20

Name three reporter genes.

Answer 20

LacZ- blue colour on X-gal
Luciferase breaks down luciferin, producing light.
GFP- excited by blue light and produces green light.

Question 21

How can we quantify expression of a gene?

Answer 21

We can tag with GFP (or similar) or express the gene over a colourimetric substrate (LacZ).

Question 22

How does western blotting work?

Answer 22

uses specific antibodies to quantify certain proteins.

• SDS is detergent used to denature the protein so it’s linear.
• PolyAcrylamide Gel Electrophoresis (PAGE) separates proteins.
• mobility depends on if it has been modified (glycosylation/phosphorylation/acetylation)
• limited by antibody availability.

Note: 2D electrophoresis of amino acids is possible as some amino acids are +vley and -vley charged. (Separates by charge and then size).

Question 23

What is the process by which proteins can be identified by mass?

Answer 23

Protein Mass Spectrometry.

-produces accurate mass readings up to 5dp!