structure and function of the bacterial RNAP Flashcards
what is meant by operons?
genes encoding enzymes that are part of the same metabolic pathway are clustered close together and regulated by the same promoter
how far apart are stacked base pairs?
3.4A
how long is a hydrogen bond?
2.8A
why does almost 100% of prokaryotic DNA code for proteins?
prokaryotic DNA is not processed and contains no introns
why is E.coli useful?
- plasmids and cloning
- model systems
- biofuels
- antibiotic production and antibiotic resistance
- no ethical issues
what are the steps in discovering an enzyme?
- make cell lysate
- invent a transcription assay; add 4NTPs, P32 ribonucleotide, DNA template, heat to 37 degrees for 30 minutes
- separate free NTP from radiolabelled NTP using ethanol
- purify using chromatography
which subunits does bacterial RNA polymerase consist of?
A-subunit, B-subunit, B’-subunit, w-subunit
what do the B and B’ subunits do?
contribute to RNA catalysis in active site
what does the antibiotic microcin do?
blocks NTP entrance channel
what is the role of the core enzyme?
synthesises RNA on a DNA template, but cannot recognise promoters
what is the role of the sigma factor?
ensure initiation of transcription from specific sites, but also reduces binding to non-specific sequences
how is the catalytic site formed?
formed by B and B’. has one Mg++ tightly bound to the enzyme in the active site, held in place by an ionic bond in coordination with oxygens and one of 3 aspartate residues
how does one promoter control synthesis of more than one protein?
there are several open reading frames
what are the steps for a transcription assay to discover an RNA polymerase?
1- make a cell lysate
2- add NTPs and a P32 labelled UTP, add a DNA template and heat to 37 degrees for half an hour
3- separate free NTP from radiolabelled NTP using ethanol
nucleic acids precipitate, but free nucleotides dont
what does the A subunit of bacterial RNA polymerase do?
responsible for enzyme assembly, promoter recognition and binding to activators
what is the role of the a and sigma subunits?
form the major surfaces on RNA polymerase for interactions of the enzyme with factors that regulate initiation of transcription
where is the channel for DNA?
at the interface of the B and B’ subunits, these stabilise the separated single strands in the transcription bubble
why does the core enzyme have general affinity for DNA?
electrostatic interactions between the protein and the DNA
what can and can’t the core enzyme do?
can: synthesise RNA on a DNA template
cant: recognise promoters
what does sigma factor do?
ensures initiation of transcription from specific sites, and reduces binding to non-specific sequences.
what is the structure of the bacterial RNAP active site?
located at the base of the cleft formed by B and B’ jaws. contains 2 catalytic Mg2+ ions, one is tightly bound to the active site, and another arrives at the active site in complex with incoming NTP
there are three aspartate residues which are located on a short loop of the beta subunit, these coordinate the Mg2+
