Prokaryotic Transcription Flashcards
What is the basic meaning of DNA transcription, and what are the main steps?
Transfer of genetic information from DNA to RNA.
Initiation - Elongation - Termination
How many RNA polymerases do PROKARYOTES have?
ONLY ONE
Discuss the features of prokaryotic RNA polymerases.
RNA polymerase synthesises RNA in a 5’ - 3’ direction
It does NOT need a primer
The RNA product does NOT remain base-paired to the template DNA strand
CODING STRAND ===
TEMPLATE STRAND ===
SENSE STRAND
ANTISENSE STRAND
How does RNA polymerase know where to start its function along the nucleotide sequence?
There are INITIATION SEQUENCES
e.g. start codons = AUG
What is the unit of transcription in prokaryotes?
Polycistronic transcripts
Outline the structure and function of the subunits of E. coli RNA polymerase holoenzyme
CORE POLYMERASE:
alpha(2)-beta-beta’-omega
beta’: core polymerase subunit (DNA binding)
beta: core polymerase subunit (polymerase active site)
alpha: scaffolding role
omega: unknown role
SIGMA FACTOR:
sigma: types, role in transcription initiation (promoter recognition), main type is sigma(70)
Briefly outline the process of INITIATION in prokaryotic transcription.
The bacterial RNA polymerase positions itself on the promoter through the sigma factor
Describe the structure of a bacterial promoter.
Sigma(70) factor;
-35 and -10 regions are instrumental in positioning the polymerase in the time leading up to initiation;
transcription start site is at +1
What is meant by ‘consensus’?
Most commonly occurring sequence out of all the successful sequences recorded for that particular gene?
The closer a promoter sequence is to the consensus…
… the stronger the promoter will be, i.e. more effective transcription; this depends on how well the polymerase is bound.
Sigma and alpha subunits recruit RNA polymerase to the promotoer; elaborate.
Sigma(2) subunit binds to -10 region –> region that melts within the active site
Sigma(4) binds to -35 region –> recognition of promoter site
UP-element: further encourages good binding of polymerase in strong promotoers, e.g. rRNA genes
Briefly outline the initiation stage of prokaryotic transcription.
INITIATION
Involves recognition and binding of RNA polymerase to the promoter (closed complex), promoter melting (open complex) and formation of a stable ternary complex (transcription bubble).
Briefly outline the elongation stage of prokaryotic transcription.
ELONGATION
Polymerase advances 3’-5’ down template strand, adding rNTPs to growing RNA chain
Briefly outline the termination stage of prokaryotic transcription.
TERMINATION
Transcription is terminated by signals within the RNA sequence; polymerase releases completed RNA and dissociates from DNA.
Define ‘terminators’
Sequences which trigger the elongating polymerase to dissociate from the DNA and release the nascent RNA.
State the two types of termination.
- Rho-dependent
2. Rho-independent
What are the features of the ‘Rho factor’ involved in Rho-dependent termination?
RHO FACTOR:
- six-ringed protein;
- binds RNA at termination recognition sites called ‘rut sites’;
- binds to single-stranded RNA;
- doesn’t bind RNA being translated;
- rho pulls the RNA out of the polymerase, or induces polymerase conformational change such that the polymerase terminates
How does Rho induce termination of transcription?
Rho pursues polymerase by moving along the growing, newly transcribed RNA strand.
A ‘hairpin’ is formed, causing polymerase to pause and allowing Rho to catch up.
The combination of protein action and destabilizing effect of hairpin loop allows Rho to induce termination
What is meant by Rho-independent termination?
Rho-independent termination requires intrinsic terminators which DO NOT involve other factors.
For example, such a terminator could include a short inverted repeat in a stretch of 8 A-T base pairs
What is the benefit of the lactose operon (lac operon)?
The lac operon allows E. coli to use lactose rather than glucose, its typical food source. It does this by hydrolyzing (breaking down) lactose into glucose and galactose.
State the name and function of each of the three genes on the lac operon.
lacZ, lacY, lacA
lacZ - produces beta-galactosidase, which hydrolyzes lactose into glucose and galacotse
lacY - produces beta-galactoside permease, which enables lactose to transport into the cell through the membrane
lacA - encodes beta-galactoside transacetylase (biological role uncertain)
Summarise transcription by RNA polymerase
INITIATION
involves recognition and binding of RNA polymerase to the promoter (closed complex), promoter melting (open complex) and formation of stable ternary complex (transcription bubble)
ELONGATION
polymerase advances 3’-5’ down template strand, adding rNTPs to growing RNA chain
TERMINATION
transcription is terminated by signals within the RNA sequence, polymerase releases completed RNA and dissociates from DNA
What is the lactose (lac) operon?
A cluster of inducible genes including: ==> lacZ, lacY, lacA (all transcribed as a single mRNA) ==> a promoter ==> a regulator ==> an operator
In what state is the lac operon normally in?
Normally, lac operon is turned off and a repressor protein binds the operator region upstream of the operon preventing RNA polymerase from reading the three lac genes.
Discuss the regulation of the lac operon.
When lactose is in the cell, lactose binds to the repressor. This causes a structural change in the repressor, causing the repressor to lose its affinity for the operator.
Thus, RNA polymerase can then bind to the promoter and transcribe the structural genes, meaning lactose acts as an effector molecule.
What are the main factors that regulate transcription in prokaryotes?
Prokaryotic transcription is mainly regulated through initiation of transcription.
Sigma factor acts as an initiation factor, and most bacterial genes are regulated by the sigma(70) factor; however, other sigma factors are available.
What is the general mechanism by which prokaryotic transcription is regulated?
The general mechanism involves a repressor that binds to the operator region of a gene or operon and blocks transcription.
The repressor is regulated through the binding of a small molecule ligand(s) which control its DNA binding activity
Some bacterial genes also respond to activators (positive regulation, e.g. CAP in lac operon).
Activators also regulated by binding of small molecule ligands (e.g. cAMP)
How many RNA polymerases are found in prokaryotes and eukaryotes?
PROKARYOTES: ONLY ONE
e.g. T. aquaticus RNA polymerase:
beta, beta’, alpha, a, psi
EUKARYOTES: FIVE
e.g. Yeast RNA polymerase II:
RPB1, RPB2, RPB3, RPB11, RPB6
For each of the eukaryotic RNA polymerases, state the name, the gene transcribed, and the sensitivity to alpha-amanitin.
RNA Polymerase I
Ribosomal RNA (45S precursor of 28S, 18s and 5.8S rRNA)
Insensitive
RNA Polymerase II
All protein-coding genes, small nuclear RNAs, U1, U2, U3, etc.
Very sensitive
RNA polymerase III
tRNA, 5S rRNA, snRNA U6, some repeated sequences (Alu)
Moderately sensitive
Mitochondrial RNA Polymerase
All mitochondrial genes
Chloroplast RNA Polymerase
All chloroplast genes
How is pre-rRNA processed?
The primary transcript is cut to yield the mature rRNAs found in ribosomes.
These are modified by methylation of the specific ribose 2’-OH.
Specific uridine (ribonucleoside of uracil) residues are then converted into pseudouridine, an isomeric form.
