Lecture 2

Question 1

What do you need to get a gene expressed?

Answer 1

• Start codon (AUG) on mRNA (+1)
• Stop codon on mRNA: ribosome stop site
• Coding region
• Promoter: RNAP binds here
• Ribosomes: machinery for translation
• Shine-Dalgarno sequence: the ribosome binding site
• Transcriptional terminator: RNAP stop site

Question 2

Methods regulation of gene expression:

Answer 2

• Repressor protein
• Activator protein
• RNA base-pairing

Question 3

Repressor protein

Answer 3

Prevents RNApol from initiating transcription

Question 4

Activator protein

Answer 4

Enhances RNA pol binding, regulating transcription initiation

Question 5

RNA base-pairing

Answer 5

RNA base pairs with RNA, influecning expression, preventing other ribosomes from binding

Question 6

Promoters

Answer 6

At the starts site of all operons, so RNA pol can initiate transcription.

Question 7

Comparative studies to understand eg. promoters

Answer 7

• Comparing promoter sequences - Shows conserved sequences at -10 and -35
• ‘Weak’ promoters tend to differ most from the consensus while ‘strong’ promoters tend to match the consensus most closely

Question 8

Consensus sequence:

Answer 8

When a number of regions of RNA are lined up a conserved sequence is visible. Variations by 1 or 2 bases are common, but most often there will be this base at this position.

Question 9

Genetic approach to understand eg. promoters

Answer 9

• Isolate mutation which affect promoter function, determine where these mutations lie in the sequence.
• Decrease in effectiveness are more common than increase in effectiveness.

Question 10

Biochemical approach to analyse protein-DNA interactions

Answer 10

• In vitro experiments using cloned fragments of DNA (eg. promoter region) and DNA-binding proteins (eg. RNAP)
• Perform Electrophoresis gel mobility shift assays
  1. Label dsDNA fragments
  2. Mobility of fragments will be slowed if the protein binds
  3. Tell you that the protein has bound somewhere..

Question 11

DNA footprinting (DNA modification protection) steps:

Answer 11

• Use to find where a protein binds in a piece of DNA.
  1. Radioactively label dsDNA on one end of one strand
  2. Isolate and denature to ss
  3. Mix with proteins
  4. Treat with DNAse1 to cleave DNA
  5. Denature to remove protein and electrophorese
  6. Use the label to visualise DNA fragments.

Question 12

DNA footprinting: how would you get a label on one end of one strand?

Answer 12

Use different types of restriction enzymes to cut a blunt end and a sticky end, then fill in using labelled nucleotides.

Question 13

DNA footprinting: what will the gel show?

Answer 13

• The control gel will show that DNA not bound to polymerase has bands at positions corresponding to breakage of every bond.
• The experimental gel will show missing bands identifying approximate binding sites, but doesn’t tell you which bases are required for binding.

Question 14

DNA footorinting: why does it matter that only one end is bound?

Answer 14

There would be two overlapping footprints, some from the top strand and some from the bottom strand. This would interfere with each other when run on a gel, and would not produce a conclusive result.

Question 15

DNA footprinting: RNAP and lac operon promoter

Answer 15

• In the -RNAP control, each band represents a DNA fragment 1 base different in length from the next. Cleavage by DNAse1 is random.
• In the +RNAP test, regions of the DNA are protected from cleavage by DNAse1.

Question 16

Positive/negative transcription

Answer 16

The function of the regulatory protein, influenced by either an inducer or repressor.

Question 17

Repressible

Answer 17

Action of small MW effector molecule eg) lactose, tryptophan. Binding prevents expression of the gene.

Question 18

Inducible

Answer 18

Action of a small MW effector molecule eg) lactose, tryptophan. Binding brings about expression of the gene.

Question 19

Cis

Answer 19

A regulatory component is at the site of the DNA, adjacent to the gene it acts on.

Question 20

Trans

Answer 20

A diffusible gene product, most likely a protein, which is expressed from one gene but acts on another gene.

Question 21

How do you determine the effect of a regulatory protein?

Answer 21

1. Mutate the regulatory gene
2. See what the effect is
3. Complement with the regulatory protein
4. See what effect this has.

Question 22

Complementation of a lacI mutant in trans:

Answer 22

• A WT chromosome produces a functional gene product, an active repressor.
• A mutant chromosome, produces inactive repressors.
• The function gene product can act in trans, so the mutation is complemented in trans

Question 23

Operator mutation lacO mutant in cis:

Answer 23

• A WT chromosome produces a functional repressor protein.
• A mutant chromosome has a mutation in the operator site, but has a functional repressor protein.
• This mutation is acting in cis, because it is a site and not a diffusible product that has mutated.
• A mutation in the operator brings about constitutive expression, the repressor can no longer bind, so the genes are always expressed.