Prokaryotic Transcription Flashcards
What is RNAP?
an enzyme that synthesises RNA using one strand of duplex DNA as a template in the 5’ to 3’ direction
What is the coding strand?
the DNA strand that has the same sequence as the mRNA
What is a transcription unit?
a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator
What is a promoter?
a region of DNA where RNAP binds to initiate transcription
What is a terminator?
a sequence of DNA that causes RNAP to terminate transcription
What is the start point (+1)?
the position on DNA corresponding to the first base incorporated into RNA (one nt downstream of the promoter)
What is the length of the transcription bubble?
~12 to 14 bp
How big is the DNA-RNA hybrid within the transcription bubble?
~8 to 9 bp
What happens as the transcription bubble progresses?
the DNA duplex reforms behind it, displacing the RNA in the form of a single polynucleotide chain
What is the transcriptional and DNA replication rate respectively?
- transcription = ~40-50 nt/s
- DNA replication = ~800 bp/s
What is nascent RNA?
an RNA chain that is still being synthesised, so that its 3’ end is paired with DNA where RNAP is elongating
What are the 3 stages of transcription?
- initiation
- elongation
- termination
How is the closed complex formed?
RNAP binding to the promoter on the DNA
How is the open complex formed?
σ factor binds to RNAP and the holoenzyme opens the DNA duplex to form the transcription bubble
What happens during elongation?
the transcription bubble moves along DNA and the RNA chain is extended in the 5’→3’ direction
What happens at termination?
the DNA duplex reforms and RNAP dissociates at a terminator
What is a holoenzyme?
the form of RNAP that initiates transcription
What are the 5 subunits of RNAP?
- 2α
- β
- β′
- ω
What is the catalytic domain of RNAP made up of?
the β and β’ subunits (the 2 largest subunits)
What do the α subunits of RNAP do?
serve as a scaffold for assembly and interacts with the promoter and some regulatory factors through its C-terminal domain (CTD)
What does the ω subunit of RNAP do?
promoter specificity
What is σ factor involved in?
the melting (unwinding) reaction; it recognises the promoter and binds to the core RNAP
What is the ternary complex?
the complex involved in initiation of transcription that consists of RNAP, DNA and nascent RNA
What does the initiation complex span?
-55 to + 20
When is σ factor released from the initiation complex?
when initiation succeeds and the nascent RNA chains reach ~10 bases in length
What does RNAP transition to after initiation?
elongation ternary complex of core RNAP-DNA-nascent RNA
How big is the elongation complex?
30-40 bases
What happens after σ factor dissociates?
the core enzyme contracts to -35 then after moving a few base pairs, it becomes more compactly organised into the general elongation complex
What does σ factor do upon interaction with the core enzyme?
change its structure to expose its DNA-binding domains
Which σ regions recognise the -10 and -35 hexamers respectively?
- -10 = 2.5 region
- -35 = 4.2 region
What does the N-terminus of σ factor do?
block the DNA-binding regions from binding to DNA
How many σ factors does E. coli have and what do they cause?
7, each of which causes RNAP to initiate at a set of promoters defined by specific –35 and –10 sequences
What is σ 70 used for?
general transcription (recognises one set of promoters)
When is a cascade of σ factors created?
when one sigma factor is required to transcribe the gene coding for the next sigma factor
What does termination require?
recognition of the terminator sequence in DNA and the formation of a hairpin structure in the RNA product
What does antitermination cause?
the enzyme to continue transcription past the terminator sequence, readthrough
What is intrinsic termination?
when the core enzyme can terminate at certain sites in the absence of any other factors
What does intrinsic termination consist of?
a GC-rich hairpin in the RNA product followed by a U-rich region where termination occurs
What must happen to form the hairpin loop?
a palindromic sequence must be formed
What is Rho factor?
a terminator protein that binds to a rut site on a nascent RNA and translocates along RNA until it reaches the RNA–DNA hybrid in RNAP
What is Rut?
the Rho utilisation site, the sequence of RNA that is recognised by the Rho termination factor
What is coupled transcription/translation in bacteria?
when ribosomes begin translating mRNA before transcription is complete
Describe bacterial mRNA
unstable with a half-life of only a few minutes
What is the 5’ UTR?
untranslated sequence upstream from the coding region of an mRNA
What is the 3’ UTR?
untranslated sequence downstream from the coding region of an mRNA
What is monocistronic mRNA?
mRNA that encodes one protein
What are polyribosomes?
a cluster of ribosomes, bound to a mRNA molecule
What does DNA footprinting do?
identify DNA-binding sites for proteins by their protection against nicking
What happens in DNA footprinting?
a bound DNA-binding protein blocks the phosphodiester bonds from attack (nuclease or chemicals), revealing the protein precise recognition site as a protected zone, or footprint
What is EMSA used for?
to study if and how a particular protein interacts with DNA
What happens in EMSA?
- when a protein binds to DNA, it will cause DNA/protein complex to move slowly through the gel
- the larger the bound protein, the greater the retardation of the DNA molecule
- gel-mobility shift assay traces the amounts of a sequence- specific DNA binding protein
