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
what percentage of RNA polymerase is actually engaged in elongation of transcription? what percentage is used in storage?
25% / 50%
what happens to DNA during transcription?
as a consequence of transcription, DNA in front of the polymerase is overwound, and the DNA behind is underwound
- two compensate, gyrase in fron to introduce negatve supercoils and topisomerase 1 and 4 are trailing to relax the supercoils
what causes most of the supercoiling in DNA?
probably results from transcription
what are the three stages of transcription?
- initiation (template recognition, closed promoter complex, open promoter complex, and abortive cycling)
- elongation
- termination
what results in the formation of the open promoter?
- irreversible and fast
- involves the melting of the DNA assisted by sigma
what is the fastest promoter clearance time? which promoter?
- ribosomal promoter is the fastest at 1 second
- still has false starts
what part of the transcript is the most unstable and what is the half life for eukaryotes and prokaryotes?
- primary transcript is the most unstable
- in prokaryotes it is degraded in 1-3.5 mins or cleaved to give mature products such as rRNA or tRNA
- in eukaryotes it is modified at the ends (mRNA 7mG CAP at 5’ end and polyA tail at 3’ end) or cleaved to give mature products (splicing or rRNA/tRNA)
how many human cells die everyday? how many blood cells die every second?
330 billion / 5 million
how long is the RNA/DNA hybrid at the active site of transcription?
9-8 nt
Phage T7 RNA polymerase
- T3 and T7 phage RNA polymerases are single polypeptides - only on unit, not multiple subunits
- they have minimal activities in recognizing a small number of phage promoters. uses a specificity loop to recognize the promoter
- contains thumbs and fingers
- promoter is very short (only 6-7 nucleotides)
- the rate of synthesis is greater than bacterial RNA polymerase at about 200 nucleotides per second
what do the crystal structure of T7 RNA polymerases identify in DNA?
- the DNA binding region
- the active site
where does the specificity loop bind to?
the T7 RNA polymerase has a specificity loop that binds to positions -7 to -11 of the promoter while positions -1 to -10 enter the active site
- the enzyme holds 10 bp upstream and 10 bp downstream from the transcription bubble
- the bases of the template strand are flipped out of the helix to be read
how large is the groove in bacterial RNA polymerase
25 angstroms wide and could hold 16 nt of DNA
- groove is positively charged but most of the enzyme is negatively charged
what is nascent RNA?
RNA that is in the process of being formed
Label all the parts
1 = beta prime
2 = main channel
3 = beta
4 = alpha 1
5 = alpha 2
6 = omega
what happens to RNA polymerase before elongation?
- it goes trough several conformational changes
- closed promoter = -55/+1
- open promoter = -55/+20 (DNA melted)
- ternary complex = (-30/+20)
how many magnesium ions do all RNA polymerases have?
2 at the active site
domain 3.2
- protrudes into the RNA exit pore and assists in the first nucleotide interaction
domain 2.3
triggers melting
domain 1.1
displaced by DNA and lies in the exit pore channel
clamp
on each side of the DNA and forms a channel
bridge
used to help incoming nucleotides in the channel
rudder
ensures RNA/DNA region is not longer than 8 nt in length
wall
- backside of the active site and DNA makes contact with it
- assists in melting and bends the DNA to facilitate the opening of the DNA
- open promoter complex
why does DNA bend when at the active site?
- helps flip the RNA away from the template strand
- the rigid structure of the double stranded DNA becomes flexible when the strands melt
- the rudder limits the length of the RNA/DNA hybrid
how does the enzyme bind the promoter without it hitting the wall?
a large conformational change must take place
- the clamp is initially out of position and repositions to keep the DNA in the active site after the DNA has melted
how does the RNA polymerase combat keeping proper positioning of the template and mascent transcript?
a protein bridge changes conformation to control the entry of nucleotides to the active site
- maintains contact with the growing strand during translocation step but snaps back once the bonds have formed
which domain of RNA polymerase prevents the RNA/DNA hybrid from getting too long?
rudder/lid
beta and beta prime
form walls of the active site
- catalytic center
alpha subunits
- controls the affinity of the holoenzyme for the promoter
- regulate frequency of initiation
- has flexible linkers
- carboxyl terminal of alpha interacts with the DNA and sees the promoter sequence in the minor groove
how large is the RNA core? with the sigma subunit?
- RNA core = 460 kD
- w/ sigma = 530
which subunit is required for promoter specificity?
sigma
what are the three roles of alpha?
- contacts DNA directly
- contact transcription regulatory proteins
what are the two outcomes of scrunching?
- sigma leaves or RNA leaves
