Flashcards in Lecture 2 Deck (23):
What do you need to get a gene expressed?
- 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
Methods regulation of gene expression:
- Repressor protein
- Activator protein
- RNA base-pairing
Prevents RNApol from initiating transcription
Enhances RNA pol binding, regulating transcription initiation
RNA base pairs with RNA, influecning expression, preventing other ribosomes from binding
At the starts site of all operons, so RNA pol can initiate transcription.
Comparative studies to understand eg. promoters
- 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
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.
Genetic approach to understand eg. promoters
- Isolate mutation which affect promoter function, determine where these mutations lie in the sequence.
- Decrease in effectiveness are more common than increase in effectiveness.
Biochemical approach to analyse protein-DNA interactions
- 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..
DNA footprinting (DNA modification protection) steps:
- 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.
DNA footprinting: how would you get a label on one end of one strand?
Use different types of restriction enzymes to cut a blunt end and a sticky end, then fill in using labelled nucleotides.
DNA footprinting: what will the gel show?
- 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.
DNA footorinting: why does it matter that only one end is bound?
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.
DNA footprinting: RNAP and lac operon promoter
- 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.
The function of the regulatory protein, influenced by either an inducer or repressor.
Action of small MW effector molecule eg) lactose, tryptophan. Binding prevents expression of the gene.
Action of a small MW effector molecule eg) lactose, tryptophan. Binding brings about expression of the gene.
A regulatory component is at the site of the DNA, adjacent to the gene it acts on.
A diffusible gene product, most likely a protein, which is expressed from one gene but acts on another gene.
How do you determine the effect of a regulatory protein?
1. Mutate the regulatory gene
2. See what the effect is
3. Complement with the regulatory protein
4. See what effect this has.
Complementation of a lacI mutant in trans:
- 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