Prokaryotic Transcription 1 Flashcards
coding strand
has the same sequence as the RNA
transcription unit
- from a single promoter
- may encode more than a single gene (polycistronic)
what is the rate of transcription?
50 nucleotides as opposed to 833 in replication
what is the stream?
- the flow of RNA polymerase
- downstream is the direction of synthesis
- upstream is the opposing, the promoter is what determines synthesis
transcription bubble
- transcription occurs by base pairing in a bubble of unpaired DNA
- the region of DNA that opens so the polymerase can read he strand
- the length of the bubble is 12-14 basepairs
- the length of the RNA-DNA hybrid is 8-9 nt
RNA-DNA hybrid
- adds nucleotides to the 5’ end
- short portion that remains as a hybrid
- RNA polymerase separates the new product from the template
- as the transcription bubble moves along the DNA template, the RNA is displaced from the RNA-DNA hybrid
what direction is RNA synthesized in?
- synthesized from 5’ - 3’
- the incoming nt loses the gamma and beta phosphate groups, leaving the alpha phosphate
what is the length of DNA-DNA at room temperature?
- short pieces of DNA easily denature
- 11 base pairs is on the border of what is stable at ambient temperatures
what is the rate of transcription?
40-50 nt per second, which is about the same rate as translation
- transcription is much slower than DNA synthesis
what is the rate of translation?
15 amino acids per second/ 45 nt of RNA per second
- as RNA emerges from the RNA polymerase, it is immediately used as substrate for ribosomes
- translation cannot be faster than transcription otherwise it would interfere with the RNA polymerase
how do ribosomes bind to RNA?
- the ribosomes scans at a ribosome binding site looking for AUG
not long after making mRNA the cell starts to degrade , why?
the cell is degraded by RNases 1.5-3 minutes after mRNA is made. This is because the cell has a short life already and the mRNA wouldn’t be needed for long
what are the proposed mechanisms for how RNA polymerase finds a promoter
- the observed rate at which RNA polymerase finds a promoter sequence is too fast to be explained by simple diffusion
- sliding
- intersegment transfer
- intrasegment transfer (hopping)
how many molecules are in an E. coli cell
13,000 molecules
where is the core RNA polymerase before transcription begins?
core RNA polymerase is stored at nonspecific DNA site before sigma binds. Core enzyme has a high intrinsic affinity for non-promoter DNA, which is increased by the presence of nascent RNA
- but its affinity for loose binding sites (non-promoter DNA) is too high to allow the enzyme to distinguish promoters efficiently from other sequences
describe the role of sigma
- when sigma subunit binds to the core of the RNA polymerase, you get a holoenzyme
- with sigma, the polymerase positive groove in the enzyme opens and loses its affinity for DNA
- when it binds to a region of DNA that is a promoter, it increases its affinity to the non specific DNA site 1000 fold
true/false: sigma decreases the affinity of the core for DNA
- cannot be answered
- causes 10,000 fold lose in affinity for nonspecific DNA
- causes 1000 fold increase in affinity for promoter DNA
what happens when the holoenzyme finds a promoter?
- forms a closed promoter complex that is reversable
- if there is not good contact, then the complex can disassociate
- only step considered to be reversible
closed promoter
DNA strands have not been melted
open promoter
- melts the two strands in order to read the template strand
- mediated by sigma subunit with a special domain
once the open promoter forms, what occurs in the holoenzyme?
- RNA polymerase does not need a primer
- RNA polymerase core tries to transcribe but often has 100-200 false starts
- once it has the first nucleotides, the subunit is still attached to the promoter and prevents transcription
- sigma subunit is stick in the RNA exit pore
- DNA scrunching of 9-8 nucleotides occurs until the sigma unit releases
ternary complex
- most stable
- protein, DNA, and RNA
- formed after the first phosphodiester bond is formed