Transcription & Control of Gene Expression Flashcards
What are the 3 types of bacterial RNA?
ribosomal (rRNA)
transfer (tRNA)
messenger (mRNA)
How do you determine long it takes for bacteria to divide with a doubling time of 20 min with 5x10^8 cells overnight?
2^n = 5x10^8 (n=intervals)
log2^n = 2log n = 5 x 10 ^8
n=17.7/0.693
=26 -> 13 hours
What does RNA polymerase need to work?
RNA synthesis needsa DNA template to work
How can we measure RNA pol activity?
Incorporate radio labelled rNTP into acid preciptable RNA
How fast and what direction does RNApol work?
transcribes 40-60 bases per second in the 5’ to 3’ direction
what are the phases of RNA transcription
initiation, elongation and termination
What are the steps of RNApol elongation?
1) Enters DNA downstream
2) DNA is unwound
3) mRNA transcribed to be complementary to the 3’ to 5’ DNA strand
4) mRNA leaves
5) RNA continues to go upstream
Describe the mechanism of the RNA polymerase catalysed reaction.
In the 5’ to 3’ direction, OH group in the nucleotide attacks the alpha Phosphorus, kicking of the pyrophosphate leaving group, forming a phosphodiester bond.
What is the role of sigma as a transcription initiation factor?
sigma factor helps RNA polymerase find promoter sequences and initiate transcription
once transcription is initiated, sigma dissociated and core enzyme continues into elongation of the transcript
How can antibiotics inhibit RNA polymerase?
Rifamycin - blocks initiation of trancription
Streptolydigin - blocks elongation of transcription
Now con you isolate antibiotic resistant mutants?
plant large numbers (10^8) of cells and use less than
optimal antibiotic concentrations
What specific changes happen to E. coli mutants to make them antibiotic resistant?
E. coli mutants were found to have identical α, β’, σ and ω RNApolymerase subunits but altered β-subunits, there is a single amino acid change, indicating β having a role initiation and elongation
What does each component of RNApol name in transcription?
β and β’ - form the catalytic centre
α and ω - required for assembly of the enzyme
α - aspects of promoter assembly
What is the process of defining transcription start sites?
A phosphate at the 5’ end of DNA is radiolabelled:
1) in vitro transcription with RNApol and rNTPs to make multiple transcripts of mRNA from the DNA template
2) denature heating
3) hybridise DNA and mRNA
4) S1 nuclease introduced, cleaving single stranded DNA (trims extra)
5) NaOH introduced to degrade mRNA as RNA is sensitive to base
6) determine DNA length to infer start site
Where and what are the bacterial promoters?
-10, pribnow box, TATAAT, essential for initiation
-35, TTGACA, controls rate of transcription
Describe the process of DNase Footprinting
A control is made where DNase cleaves the phosphorilated DNA at various positions, creating multiple fragments, observed via denaturing gel electrophoresis and audioradiography.
The same is run but with RNApol, and the ~70 base footprint region can be seen by comparing the missing fragments to the control, as RNApol binding obscured DNase cleavage
How can the lack of -35 region of promoter effect mRNA synthesis?
when observed with exonuclease, the DNA is degraded from the ends and -35 removed, a short mRNA transcript is produced but RNApol cant rebind as -35 required for binding but not initiation.
Describe the stem-hairpin loop structure
a palindromic sequence that runs in opposite directions and when folded into a hairpin loop, one side of the stem is complimentary to the other
it is a rho-independent terminator
has a G:C rich stem
is followed by a run of U’s (UUUUUU)
describe Rho-idependent termination
1) hairpin loop in mRNA causes RNApol to pause
2) weak dA-rU DNA-mRNA base pairing causes thermodynamic dissociation
3) RNA dissociates from DNA
4) mRNA dissociates from RNApol
5) DNA strands recombine as transcription bubble closes
When is Rho-dependent termination needed?
When mRNA is not followed be a run if U’s
What is Rho and Describe Rho-dependent termination
Rho is a protein of 46 kDa and is hexameric
It binds to RNA at rut (rho utilisation) sequences and tracks along mRNA in the 5’ to 3’ direction
Its helicase activity separates mRNA from the DNA template
Define constitutive gene expression
Genes whose levels of transcription are determined solely by the strength of the interaction of RNA polymerase with the promoter (K) are said to be constitutively expressed
How do E. coli utilise lactose
When lactose is introduced to the cell, lacZ (beta galactosidase) rises 1000 fold.
lactose can then be hydrolysed into galactose and glucose or isomerised into allolactose
Describe the lac operon
First there is the promoter followed by short (38 kDa) transcribed lacI (lac repressor) which is then terminated
Then there is a second promoter and then operator which the transcribes 3 proteins of lac mRNA:
lacZ - beta-galactosidase (125 kDa)
lacY - lactose permease (30 kDa)
lacA - beta-galactoside transacetylase (30 kDA)
What is the half life (t 1/2) of lac mRNA?
3 minutes so the system can respond quickly to appearance or depletion of lactose
Is lactose a beta-galactosidase substrate or inducer?
Substrate
Is isopropyl-beta-D-thiogalactose (IPTG) a beta-galactosidase substrate or inducer?
inducer
Describe the DNase footprinting of LacI (lac repressor)
all DNA marked at 5’ end with radiolabelled phosphate
i) control of lacPODNA and DNase, breaks into fragments which are observed with denaturing gel electrophoresis
ii) lacPODNA + DNase + LacI (lac repressor)
iii) lacPODNA + DNase + lacI + IPTG
isopropyl - beta - D - thiogalactose (IPTG) prevents cleavage
when all fragments compared, the missing section in ii shows the footprint of lacI (lac repressor)
What is the lac control region?
The overlap of RNA polymerase and lacI (lac repressor)
in this space, genes are switched off until the introduction if lactose
How can you identify the lac operator?
there is a palindromic sequence called the “synthetic olgionucleotide duplex”
How does the lac operator structure help its function?
it has looping which allows lac repressor to bind in multiple places
How do the concentrations of glucose and cAMP in an E. coli cell relate to each other?
they are inversely correlated (when one goes up the other goes down)
Where is the activator site in the lac control region?
Upstream of RNA polymerase as it promotes the process
What 2 things are required for lac operon expression?
1) a positive stimulus by Crp
2) the absence if a negative stimulus from lacI (lac repressor)
What is the role if Crp?
it binds to cAMP, this complex then can bind to the activator site
it also binds to other DNA sections to prevent any sugar other than glucose
How do the sugars communicate to RNA polymerase?
through receptor proteins
How can alternate sigma factors be useful?
they can recombine with core RNA polymerase and direct to distinct sets of promoters that they have the ability to recognise,, this then allows a new set of genes to be transcribed.
this is good for when large sets of genes that are only needed in particular physiological circumstances
What are the 2 alternate lifestyles of bacteriophage λ?
lysis and lysogeny
What does lysis require?
replication of phage particles i.e. viral proteins and DNA and rupturing of the host cell membrane
What does lysogeny require?
requires the prevention of replication of phage particles, instead needs integration of the phage DNA into the bacterial chromosomal DNA
What is the cos site?
“Cohesive” - has single strands on each end
what is cI?
λ repressor. acts as a repressor at P(L) and P(R), and an activator at P(RM)
-turns off early genes, turns on its own gene
what is cII?
activator of transcription from P(RE) and P(l), there is no transcription from these promoter unless cII is present
-turns on cI recombination genes
what is cIII?
acts as a protector if cII, enhancing its stability from degradation
what is Cro?
a repressor at P(RM)
as Cro concentration rises, it binds to and represses transcription at P(L)
binds to P(R) to turn off its transcription
what is N?
an antiterminator of transcripts initiated at P(L) and P(R)
- turns on cII, replication, Q, cIII and recombination genes (early genes)
how do rich vs starved conditions effect if lysis or lysogeny is followed?
in rich conditions -
- cells grow, proteases active -> follows lysis
- new hosts available -> cII is degraded
- no cI from P(RE)
in starved conditions -
- cells not growing so no new hosts -> follow lysogeny
- absence if proteases -> cII is stable and active
- cI made from P(RE)
How do cI and Cro bind to O(R) sites?
Cro-
has 10 fold higher affinity for OR3 than OR2 and OR1 sites
cI-
has 10 fold higher affinity for OR1 than OR2 and OR3
they fill the sites oppositely so that each first turns the others gene OFF
What is special about cI binding and Cro binding?
they are cooperative (looks like theyre hugging)
what us the function of lexA?
represses genes required to repair DNA in the SOS response
