Prokaryotic Transcription Flashcards
Which direction does transcription take place?
5’ to 3’
What is the coding strand?
DNA strand that has same sequence as mRNA
Define promoter
Region of DNA where RNAp binds to initiate transcription
Define terminator
Sequence of DNA that causes RNAp to terminate transcription
Define transcription unit
Sequence between site of initiation and termination by RNAp
May include more than one gene
What is the start point?
+1
Position on DNA where the first base is incorporated into RNA
What happens during transcription?
Separates DNA strands
Transient transcription bubble
Template strand
DNA duplex reforms behind bubble = displacing RNA
How is the bubble maintained?
Bubble is maintained within bacterial RNA polymerase
Define nascent RNA
RNA chain that is still being synthesized
Its 3’ end is still paired with DNA where RNAp is elongating
What happens in initiation of transcription?
RNAp binds promoter on DNA = forms CLOSED complex
RNAp initiates transcription after opening DNA duplex to form transcription bubble = OPEN complex
What happens in elongation of transcription?
Transcription bubble moves along DNA and RNA chain is extended 5’ to 3’ direction
What happens in termination of transcription?
Transcription stop
DNA duplex reforms
RNAp dissociates at a terminator
Why does abortive initiation occur?
Suboptimal promoter sequences, such as weak or non-consensus sequences
Low availability and activity of initiation factors, such as sigma factors in bacteria or general transcription factors in eukaryotes
Regions with high GC content or secondary structures may hinder the progression of RNA polymerase and promote abortive initiation
Availability of nucleotides
How does abortive initiation stop?
What is a holoenzyme?
A fully active form of an enzyme
What subunits are part of the RNAp holoenzyme?
Core enzyme with 5 subunits = 2 alpha, beta, beta’ and omega
And a sigma factor
What do the beta and beta’ subunits do?
Catalytic centre
Make up most of enzyme mass
What is the role of alpha subunit?
Scaffold for assembly
Interacts with promoter and regulatory factors through its CTD
What is the role of sigma factor?
Promoter specificity
Melting/unwinding
What does RNAp closed complex mean?
When RNAp binds the promoter = DNA remains double stranded
What is the ternary complex?
Initiation of transcription complex
RNAp, RNA and nascent RNA
When is the elongation complex formed?
When initiation succeeds and nascent RNA chains ~ 10 bases
Sigma factor is released
Elongation ternary complex is formed
What happens when the sigma factor dissociates?
Core enzyme contracts to -35 then after a few more bases = compacts into general elongation complex
Why does sigma factor change its structure?
To expose its DNA-binding regions when it associates with core enzyme
What is the UP element?
Sequence in bacteria next to promoter = enhances transcription
Upstream of -35 element
What is the structure of sigma factor?
Sigma 2 & 4 = DNA-binding domain
N terminus blocks binding region when not associated with core enzyme
DNA displaces N-terminus when complex forms
What is the promoter sequence?
Idealized sequence, where each position represents the base most found when sequences are compared
What does the promoter sequence consist of?
Purine at start point
Hexamer TATAAT at -10
Hexamer TTGACA at -35
Do E.coli have different sigma factors?
Yes, 7 sigma factors = each causing RNAp to initiate a set of promoters defined by specific -35 and -10 sequences
What are the differences between the sigma factors?
Induced by particular environmental conditions
Recognize promoters with different consensus sequences
Different uses and genes encoding them
What is sigma factor 70 used for?
General transcription
When is a cascade of sigma factors created?
When one sigma factor is required to transcribe the gene coding for the next sigma factor
How is termination induced?
Termination requires recognition of terminator sequence in DNA
AND the formation of a hairpin structure in the RNA product
What does antitermination cause?
Readthrough = enzyme to continue transcription past the terminator seqeunce
What is intrinsic termination?
Core enzyme can terminate at certain sites
In absence of other factors
What does intrinsic termination require?
G-C rich hairpin in RNA product
U-rich region where termination occurs
Why does the hairpin need G-C rich region?
G-C is stronger = need a stable stem otherwise termination would be compromised
What is needed in Rho-dependent terimination?
Rho factor = terminator protein
rut site on nascent RNA
What is the rut site?
rho utilization site
Where Rho factor bind to RNA and translocates along RNA until it hits RNAp
How do transcription and translation occur in bacteria?
Simultaneously = they are coupled
mRNA is transcribed, translated, and degraded simultaneously in bacteria
What is bacterial mRNA properties?
Unstable
Half-life of a few minutes
As opposed to in eukaryotes = RNA relatively stable and continued to translate for several hours
What regions do bacterial mRNA include?
Untranslated and translated regions
What do 5’UTR and 3’UTR stand for?
5’ = untranslated sequence upstream from coding region of mRNA
3’ = untranslated sequence downstream from coding region
What is a monocistronic mRNA?
mRNA that encodes one protein
What is the intercistronic distance?
When start and stop codons overlap
This can occur because of different reading frames in prokaryotes
Does the intercistronic distance vary?
Yes, varies from -1 to +40 bases
What does it mean for bacterial mRNA to be polycistronic?
Having several coding regions that represent different cistrons
What is electrophoresis-mobility shift assay used for?
Te detect protein-DNA interactions
What is immunoprecipitation used for?
To isolate and concentrate a particular protein from a mixture of many different proteins
What is DNA footprinting used for?
To locate a protein binding site on a particular DNA molecule
