11 - Bacterial Transcription Flashcards
which strands are ‘sense’ and ‘nonsense’ in bacterial transcription
5’ - 3’ - non-template ‘sense’ strand
3’ - 5’ - template strand ‘antisense’ strand
Difference in base pairing in RNA synthesis in bacteria and DNA synthesis in eukaryotes
RNA synthesis in bacteria contains Uracil as a base instead of Thymine
- othewise, follows same base-pairing rules as DNA
Why is uracil not found in DNA
Any uracil produced by spontaneous deamination of cytosine can cause mutations in daughter strands
- so is removed
How is uracil produced as a base in DNA
- why is this bad?
Cytosine bases in DNA replication can undergo spontaneous deamination to produce uracil
- can lead to mutations if this occurs, as a mutant daughter strand
How is uracil removed as a base in DNA replication, and how is DNA fixed after
- U is removed by uracil-DNA glycolsylase
- generates an abasic site
- absasic site is removed and repaired by DNA polymerases
3 major classes of bacterial RNA and functions
mRNA - encodes proteins
rRNA - constituents of ribosomes: role in translation and protein synthesis
tRNA - adaptors between mRNA and amino acids: role in protein synthesis
RNA Polymerase function in E. coli
- how differs from eukaryotes
synthesises the 3 major classes of bacterial RNA (mRNA, rRNA, tRNA)
- in eukaryotes, there is separate RNA polymerase for each class
advantage of bacteria having no nucleus in transcription and translation
allows transcription and translation to occur simultaneously
bacterial transcription unit
often an operon
Function of different parts of operon in bacterial transcription
5’ promoter - attracts and binds RNA polymerase - expect an operator
Protein coding (transcribed) sequences - often multiple genes (polycistronic) - part of operon
3’ terminator region - signals stop point for transcription
structure of Bacterial RNA polymerase
- how to convert to holoenzyme
bacterial core RNA polymerase made of multiple subunits:
- alpha
- Beta
- Beta’
- w (omega) subunits
- in ratio of 2:1:1:1
- addition of a sigma subunit converts enzyme to a holoenzyme
RNA Polymerase binding to DNA at promoter sequences
- core RNA polymerase binds DNA non-specifically and can slide
- sigma subunit binds to core polymerase to produce holoenzyme
- directs holoenzyme to a gene promoter to transcribe DNA
Method to isolate promoter region in operon in bacteria
- what this method forms basis for
- bind RNA polymerase holoenzyme to DNA in vitro
- Add nuclease
- DNA is degraded, except for stretch bound to polymerase, which is protected
- forms basis of DNA footprinting technique used to identify promoters
regions on bacterial transcription units that are protected by RNA polymerase
- 2 promoter regions
- both near start of transcription (-10 sequence and -35 sequence from start)
- both sites have conserved bases (e.g. TTGACA) and are shown in most promoter regions
directionality of promoter sequence
- asymmetry of promoter region provides directionality
- -10 and -35 start sequencea are defined on SENSE (non-template, non-transcribed) strand
- RNA built in 5’ to 3’ direction
- new nucleotides added at 3’ end
- using antisense strand as a template (GC, AU)
3 stages of transcription
- brief summary of each
- initiation - RNA polymerase holoenzyme binds promoter, opens DNA double helix, and starts to transcribe
- elongation - sigma subunit disengages from holoeznyme, core enzyme continues to make new RNA
- termination - RNA polymerase core enzyme dissociates from DNA, transcription halts
Transcription of bacterial DNA - initiation mechanism
- core polymerase consists of 2 alpha, 1 B, 1 B’ and 1 omega subunit
- the core DNA polymerase binds DNA non-specifically and can slide
- An alpha subunit binds to the core polymerase and directs the polymerase holoenzyme to a promoter
- holoenzyme binds to the -10 and -35 regions
- there are multiple alpha factors, which can bind to different promoter regions - specificity
Scrunching, abortive initiation and success:
- polymerase pulls downstream DNA towards itself
- scrunching the DNA
- until success, where -10 region is opened
- this converts closed promoter complex to an open promoter complex - does not require ATP hydrolysis (unlike DNA helicase)
- 12 to 15 bp are unwound from within the -10 region to position to +2 or +3 - transcriptional start site is exposed
- RNA polymerase makes RNA copy from the template strand using bp rules (CG, UA)
- RNA polymerase does not require a primer (unlike DNA Pol)
- After around 10 nucleotides of RNA synthesis, alpha factor is now exposed and disengages
- RNA polymerase can now elongate the new RNA - transcriptional elongation now occurs
Transcription - elongation mechanism
- transcription ‘bubble’ forms for RNA Pol function
- during elongation, RNA polymerase is highly processive of DNA strands - DNA is processed quickly through the RNA pol (polymerase) core to produce RNA
proofreading:
- if RNA Pol mis-incorporates a ribonucleotide
- it hesitates and then back-tracks to remove the nucleotide
- then continues transcrip
- Error rate: 1 mistake every 10^4 - 10^5 nucleotides, even with proof-reading
Transcription - termination mechanism
2 mechanisms:
- Rho (p)-independent - terminator sequence in RNA is recognised
- Rho (p)-dependent - requires Rho (p) protein to break the RNA:DNA duplex in transcription
- In both cases, the functioning signals are recognised not in the DNA template, but in the newly synthesised RNA.
Rho (p)-independent:
- DNA encodes STOP signals for transcription
- simplest encoding is a GC rich sequence, followed by a T rich sequence
- transcription terminates in this run of Ts or just after them
Rho (p)-dependent termination:
- Rho (p) protein is a hexameric helicase that binds a C-rich, G-poor sequence in RNA
- uses its helicase activity to chase RNA pol
- it then caches
- this disrupts the DNA:RNA hybrid helix
- releasing the RNA
what provides directionality to bacterial DNA
asymmetry of the promoter sequence
how specificity provided for different promoters in bacterial transcription
- different alpha subunits and factors recognise different promoters and provide specificity
Rate of transcriptional elongation in bacteria compared to eukaryotes (with DNA Pol)
bacteria have slow elongation compared to DNA Pol in eukaryotes
Rate of transcriptional elongation in bacteria (RNA Pol) compared to eukaryotes (with DNA Pol)
bacteria have slow elongation in RNA Pol compared to DNA Pol in eukaryotes
two transcriptional termination mechanisms in prokaryotes
Rho (p)-independent - a terminator sequence in RNA is recognised
Rho (p)-dependent - requires Rho (p) protein to break the RNA:DNA duplex in transcriptional bubble
why such a high error rate in prokaryotic RNA transcription elongation
- DNA errors are transmitted to progeny cells: they are stringently repaired.
- RNA errors mean that some transcripts may be mutated, but the majority are not.
- If the transcript encodes a protein, them most of that protein will be fine, but a small subpopulation may be mutant – and can probably be tolerated.
prokaryotic transcription inhibition
- Rifampicin is an inhibitor
- inhibits RNA Pol by binding tightly to RNA exit channel
- so affects initiation (but nor RNA Pol at elongation stage)
RNA Pol bending DNA duplex use
allows the duplex to be opened up more easily
