EC2: RNA polymerase, RNA processing, and antibiotics & RNA polymerase Flashcards
What are the different sections of bacterial RNAP, and how many of each are there?
- 2 x α (alpha)
- 2 x β (beta)
- 1 x ω (omega)
What is a holoenzyme?
What is RNAP’s holoenzyme?
A core enzyme in its complete functional form, plus additional components
For RNAP: core plus sigma (σ)
How do RNAP’s core and holoenzymes differ in function?
-
Core enzyme:
- Native DNA: random starts
- Foreign DNA: random starts
-
Holoenzyme:
- Native DNA: specific starts (highly selective)
- Foreign DNA: random starts
What does the sigma region do in RNAP’s holoenzyme?
It recognises specific promoters, but only from within the holoenzyme
How do RNAP’s holo- and core enzymes work together in transcription?
The holoenzyme recognises the promoter and initiates transcription
The core enzyme elongates and completes transcription
What are the functions of RNAP’s subunits?
- β and β’: active site processivity*
- the active site is shared between the β-subunits when they come together
- α: DNA binding (non-specific)
- ω: enzyme assembly (only recently confirmed)
- it is part of the glue that holds the holoenzyme together
- σ: promoter recognition
*processivity = enzyme’s ability to catalyze consecutive reactions without releasing its substrate
What is the role of sigma factors?
They regulate gene expression in bacteria:
- They get turned on and off
- The presence of sigma factors turns on blocks of genes when needed
- When they are destroyed, families of genes are turned off
- They are highly specific
Describe the different stages of bacterial transcription.
- Initiation: the holoenzyme opens the promoter – a region of about 2 helical turns – and forms a transcription bubble
- Elongation of transcription: the bubble advances along the RNA, with DNA being unwound ahead of it. Nascent RNA is made as a complementary copy of the template strand. DNA is rewound behind the bubble.
- Termination of transcription: (in bacteria) sometimes dependedent on rho factor (ρ), sometimes not. Uses energy from ATP hydrolysis
Do RNA polymerases need a primer?
No – they work de novo
What is at the 5’ end of the nascent RNA during the elongation of transcription?
A triphosphate (ppp)
What does the triphosphate at the 5’ end of nacent RNA act as?
A marker for the intact, 5’ end of nascent RNA
What is the angle fo the bend in DNA in the transcription bubble?
90° (right angle)
What is present in the RNAP core enzyme to keep the DNA strands apart once they have been unwound?
Knobs and valleys
How do the substrates get into RNAP core enzyme?
What ion does this use?
Through the NTP channel, which uses Mg2+
What is another name from post-transcriptional modification of RNA?
RNA processing
What proportion of genes within a cell are to do with RNA processing?
Approx. 10%
Are both eukaryotic and bacterial mRNA processed?
No, only eukaryotic
What are the two different types of rRNA/tRNA processing?
- Chemical modification
- Cleavage and trimming
What are the three different types of chemical modification of rRNA and tRNA?
- Base modification
- Ribose methylation
- Pseudouridine formation
What is the simplest form of base modification of rRNA/tRNA?
N-methyl
How does pseudouridine (pseudo-U / Ψ) formation work?
A ribose is added to the 5’-carbon of a uricil base rather than the 1’-carbon, meaning nitrogen is now a proton donor and can form hydrogen bonds
What is the primary transcript?
The precursor to tRNA
Where do all chemical modifications happen?
On the primary transcript
What are mature rRNAs?
Pre-rRNA with all chemical modifications complete and introns spliced out
What are the three types of eukaryotic RNAP and what are their functions?
- RNA pol I: makes larger rRNAs
- RNA pol II: makes mRNAs
- RNA pol III: makes tRNAs and small RNAs, e.g. in ribosomes
Where are each of the eukaryotic RNAPs found?
- RNA pol I: in the nucleolus
- RNA pol II & III: in the nucleoplasm
How many subunits are in each of the eukaryotic RNAPs and the bacterial RNAP?
- RNA pol I: 14
- RNA pol II: 12
- RNA pol III: 17
- bacterial RNAP: 5
How do the drugs rifamycin and its chemical derivative, rifampicin, work? Against which disease are they used?
They are used against tuberculosis (TB)
They bind to the β subunit of bacterial RNAP and inhibit transcription
Note: they have no effect on eukaryotic RNAPs
Against which eukaryotic RNAPs is alpha-amanitin effective? Where in nature is this drug found?
RNA pol II in the liver and slightly against RNA pol III
Amanita phalloides a.k.a. Death Cap
