Lecture 3 - Transcription machinery: RNA polymerase and regulatory sequences Flashcards
What are the basic features of transcription?
- uses dsDNA
- template strand and coding strand
- RNA transcript complimentary to the template (T replaced with U)
- RNA polymerase
- RNA polymerase binds the promotor and moves along transcribing until it reaches the terminator
- transcription unit can contain a single gene or man y
What is defined as a transcription unit?
the sequence of DNA transcribed into a single mRNA, starting at the promotoer and ending at the terminator
What does RNA polymerase do?
- separates/unwindsw the 2 strands of DNA to form a transcription bubble that is around 12-14bp long (DNA must be denatured for this to be acheived)
- RNA pol moves in the 5’ to 3’ direction along the template strand
- as it moves downstream DNA is annealed behind it
How does RNA pol synthesise the RNA chains?
- RNA is synthesised by complimentary base pairing with the template strand of DNA
- RNA pol catalyses the formation of a phosphodiester bond between 2 bases
- OH of C3 on the adenosine transcript binds with an alpha p on the other nucleotide (cytidine triphosphate) through nucleophilic attack
What are the differences in RNA pol between bacteria, archaea and eukaryes?
-bacterial polymerases have the least subunits and is least complex, eukaryotic pol has most
Structure
-eukaryotic pol has 3 polymerases - I, II, III
-eukaryotic pol are largest
Associated factors
-bacterial pol has all it needs to bind DNA
-eukary and archaea require associated initiation and elongation factors
-eukaryotes require the most associated factors
What are the features of the bacterial RNA polymerase?
- 2 multisubunit enzyme
- contains an internal channel/groove (probably where DNA is accomadated)
- length of the groove can hold 16bp of DNA
- groove is lined with positive charges (bind to DNA, slightly negative)
- exists in two forms: holoenzyme and core
- holoenzyme bound to a σ factor which enables the holoenzyme to bind specifically to promotor sites
- Core made up of: 2 α subunits, β, β’, ω
What are the genes that encode the 5 bacterial RNA pol subunits and what are their features?
α: (40kD)
-rpoA gene
-enzyme assembly, recognition of UP-suqences at the promoter, binding of some activators
β: (155kD)
-rpoB gene
- part of the catalytic site, binding DNA and RNA
β’: (160kD)
-rpoC gene
- part of the catalytic site, binding DNA and RNA
σ:
-rpoD
-promotor specificity, involved in initiation not in elongation
ω:
-rpoZ
-enzyme assembly
What is the action of Rifampicin?
- antibiotic for the treatment of mycobacterium infections (leprosy, tb) isolated from a soil bacterium
- attacks the β subunit
- stops enlongation of bacterial genomes by blocking the b subunit
What are the stages of the transcription reaction?
Initiation
-includes all the steps of transcription up to synthesis of the 1st bond in RNA
-includes RNA pol finding the promoter, binding of RNA pol to the promoter, melting of DNA to form the transcription bubble
Elongation
-stage during which RNA transcript is extended by the addition of ribonucleotides
-rate= 40nt/s (very slow comp to DNA polymerase 800nt/s)
Termination
-end transcription by stopping the addition of ribonucleases
-causes dissociation of RNA pol from DNA and releasing of the RNA transcript
What is the mechanism of initation?
- Holoenzyme binds DNA in a closed binary complex (DNA still annealed) at the promoter 75-80bp of DNA contained by the complex
- DNA melting(sigma2) - now an open binary complex through the helicase activity of the polymerase forming a transcription bubble 14-16 bp long
- Ternary complex - made up of DNA, RNA and RNA pol begins abortive initiation (tendency of bacterial RNA polymerase to release mRNA transcript as it transcribes through the promoter producing truncated transcripts)
- Promoter clearance and the start of elongation, at which point σ may be released
What is the promoter consensus sequence?
-Sequences upstream of all genes to be transcribed
-consensus sequence constructed by aligning all known sequences to define which base is most predominant at each position of the sequence
-may contain A-T rich element (UP element): located upstream of the -35 and contacted by the α subunits of RNA pol
Conserved features of the bacterial promoter:
-35 TTGACA and -10 TATAA box: both 6bp motifs normally at the same positions (96% of promoter have a T at the end of -10 site)
-start point = A/G at +1 (90% of the time it is a purine base)
-spacing between the -35 box and the -10 box
What are the different regions of the sigma factors and what are their functions?
Functions:
-promoter recognition
-promoter melting
-to decrease the affinity of RNA pol holoenzyme to random sequences
Regions
-σ1.1 (autoinhibition), σ2 (promoter melting) σ3 and σ4 (promoter recognition and binding, σ3 for -10 box, σ4 for the -35box)
-when unbound to core enzyme, sigma factor cannot bind DNA alone
-N terminal region (σ1.1) binds to the DNA binding domains leading to autoinhibition
What is the structure of sigma factors?
- have an elongated shape
- DNA binding domain in the C terminus
- σ1.1 (autoinhibition), σ2 (promoter melting) σ3 and σ4 (promoter recognition and binding, σ3 for -10 box, σ4 for the -35box)
How do sigma factors work?
Before σ2 binding
-no access to the base contents
-no promoter melting and no transcription
After interaction with the σ2 region
-causes bases to ‘flip out’ and allow a direct readout of the base contents, promoter melting and transcription
Give some examples of some alternative sigma factors, their genes and their functions
σ70: (primary sigma factor in E.coli) -rpoD -general/growth related -1000 genes under control σs: -rpoS -stress response -~100 genes under control σ32: -rpoH -heat shock -~40 σE: -rpoE -extreme heat shock -~5 σ54: -rpoN -nitrogen starvation -~15 σ28: -pIIA -flagella synthesis -~40
