Transcription Flashcards
What polymerase is used in prokaryotic transcription?
RNAP holoenzyme 449 kDa, 6 subunits total
Core: α2ββ′ω
The σ subunit dissociates when RNA synthesis initiates, which carries out the polymerisation process
It doesn’t require a primer
Mg2+ and Zn2+ metal ions
Open complex - crab claw, with two pincers of the β and β′ subunits (27 Å)
Closed complex - 10 Å, formed when RNAP holoenzyme is in complex with dsDNA containing a promotor
Inner surface - positive
Outer surface - negative
What are the roles of the subunits of RNA polymerase?
α2 - N-terminus domain (NTD) - assembly of RNA pol
C-terminus domain (CTD) - non specific interact with promotor
β′ - active site for RNA synthesis
β - involved in RNA synthesis
ω - assembly of RNA pol and stabilises RNA pol
σ factor - allows the specific binding of RNA pol with the promotor
What portion of DNA is transcribed within prokaryotic DNA?
The antisense or non-coding strand is transcribed as the complementary transcribed sequence of RNA will be same as the coding/sense strand
Protein coding genes in prokaryotes - operons
Operons are multiple genes arranged in tandem along a single DNA strand so they are transcribed together
Sigma factors control gene expression:
Early/middle/late genes
Describe initiation P1 of prokaryotic RNA transcription?
Closed complex:
RNAP binds to a promotor region in dsDNA form
Promotor - 40 bp, upstream of the transcription site (- value), it commonly interacts with -10 and -35
The dsDNA starts to unwind/melt forming a transciption bubble
Describe a potential initiation P2 of prokaryotic RNA transcription?
Abortive initiation
In the open complex RNAP starts adding rNTPs (no primer required)
RNA pol starts to form short RNA stretches 9-11 nt, which are continually released
This occurs because the sigma factor blocks the RNA exit channel on the RNA pol
RNAP fails to escape the promotor, relieving the conformational tension by releasing RNA and the transcriptional bubble can relax
Describe elongation of prokaryotic RNA transcription?
The sigma factor dissociates allowing the core enzyme to tightly bind (with high processivity)
RNA synthesis grows in the 5’ to 3’ direction with RNAP moving along the template strand (non-coding strand)
DNA is continuously unwound, breaking H bonds ahead of the enzyme (positively supercoiled)
DNA is rewound behind the core enzyme as H bonds are reformed (negatively supercoiled)
The DNA-RNA bp aren’t very stable but RNAP acts as a linker whilst in elongation
Describe one type of termination of prokaryotic RNA transcription?
Two types: Rho independent and Rho dependent
Rho Independent - No external proteins required
2 elements are utilised:
1. Inverted repeats
2. 8 A:T bp (AT rich sequence)
When the repeats are transcribed - RNA forms aq stem loop hairpin, halting the RNA pol
The following A:U bp transcribed are the weakest bp
Therefore RNA is released ending transcription
Describe the other type of termination of prokaryotic RNA transcription?
Rho dependent termination:
Rho protein = hexameric ATP dependent helicase, 419 residues
It binds ssRNA rich in C and poor in G (called rho utilisation site = Rut site)
Rho sensitive pause site = 100 nt away from Rut, where RNAP is paused
Rho uses ATP to unwind RNA-DNA double helices by translocating along ssRNA in the 5’ to 3’ direction - overtaking RNAP
During translocation it moves one nucleotide along the RNA for every ATP is hydrolyses (due to 6 subunits successively undergoing hydrolysis)
What is significant about eukaryotic RNA transcription?
Eukaryotic transcription has multiple RNAPs and a more complicated control sequence and machinary
The polymerases require general transcriptional factors to recruit and position the polymerase at the promotor
What are the eukaryotic RNA polymerases?
RNA pol I - synthesises the precursors for rRNAs
RNA pol II - synthesises the precursors for mRNA
It binds 2Mg2+ at the active site
RNA pol III - synthesises the precursors for tRNA and 5S rRNA
All contain 2 nonidentical large subunits and 12 different small subunits
Many are homologs to the subunits in prokaryotes and are highly conserved
Describe the promotor region in eukaryotes?
It is very long
Structural genes expressed in all tissues (housekeeping genes) - GC-rich DNA
Structural genes that are selectively expressed - lack GC-rich sequences, they contain a core promotor 40-50 nt
e.g. TATA box (AT-rich) located 25 to 31 bp upstream of the transcription start site
Describe initiation P1 of eukaryotic RNA transcription?
TFIID binds to the TATA box promotor region, TFIID contains a TBP - TATA binding protein
TBP bends the DNA by 45° in between the first 2 and last 2 bp of the TATA box
TBP uses H bonds and van der Waals interactions
The kink is stabilised by two Phe side chains, flanking each kink from their minor groove sides
The bent conformation creates a stage for assembly of other proteins - to form the Pre-initiation complex
TFIIA - binds and helps stabilise the binding of TFIID with the promotor
Describe initiation P2 of eukaryotic RNA transcription?
TFIIB - binds and interacts with TBP & the promotor region (downstream to the TATA sequence) - helping the recruitment of RNA Pol II
TFIIF - binds to RNAP II and helps escort/recruit it to the promotor region, binding to TFIIB
TFIIE - binds to the transcription complex, helping with the binding of TFIIH
TFIIH - binds
= Pre-initiation complex (PIC)
Describe TFIIH?
TFIIH (large 9 subunits) - 2 subunits have ATPase activity - acting like a helicase, melting the promotor to induce the open complex formation
7 subunits have kinase activity to phosphorylate the CTD of RNA pol II in order to escape the promotor and start elongation
Describe elongation of eukaryotic RNA transcription?
RNA pol II is phosphorylated this stimulates elongation
TFEb (kinase protein) - phosphorylates serine residues in the CTD of RNA pol II
Serine 5 -> allows release of PIC
Serine 2 -> conformational change in RNA pol II to elongate
TFIIS - helps polymerase overcome transcription blocks and helps increase the rate of transcription where slow
mRNA is produced in the 5’ to 5’ direction and 5’ capping takes place
What are the protective measures that take place on mRNA?
5’ cap protects mRNAs from 5’ degradation and facilitates translation (bound elongation factor)
The polyA tail protects mRNA from 3’ degradation - helps export mRNA from the nucleus and facilitates translations
Splicing provides opportunities for protein diversity in tissue, development etc…
Describe termination of eukaryotic RNA transcription?
There is no defined termination sites for RNA polymerase II
It stops at various distances downstream of the protein-coding gene
At the end the protective measures take place
How is the 5’ cap formed?
Takes place whilst RNA is being transcribed (soon after initiation), around 30 nt long
At the 5’ end of mRNAs:
1. RNA triphosphatase removes a gamma phosphate
2. Guanylyation by a guanylyltransferase adds a guanidine cap (uses GTP), forming 5’-5’ triphosphate and PPi
3. Guanine is methylated by guanine-7-methyltransferase, using SAM to supply the methyl group
How is the polyA tail formed?
CstF and CPSF are recruited to the CTD of RNA pol II
CPSF = Cleavage and Polyadenylation Specificity Factor - binds AAUAAA on RNA
CstF = Cleavage Stimulation Factor - binds a GU-rich sequence downstream of AAUAAA
CstF cleaves the mRNA and the dissociates
CPSF then recruits poly A polymerase assing 200 adenine residues at the 3’ end (using ATP) = poly A tail
Poly A binding protein binds to the polyA tail and CPSF is releasesd
PolyA binding protein prevents degredation of the polyA tail
What mature mRNAs lack a polyA tail?
Histone mRNAs
They have a protective hairpin to stop the degradation of the mRNA
What does splicing do?
Removes introns from eukaryotic genes and splices together the exons from pre-mRNA or heterogenous nuclear RNAs (hnRNAs)
pre-mRNA contains 8 introns on average and are 4-10x largers than exons (average 1800 nt)
How does splicing occur?
The splicing occurs via two transesterification reactions
1. A 2′,5′-phosphodiester bond forms between an intron adenosine residue and the intron’s 5′-terminal phosphate group
2. The 5′ exon is released and the intron forms a lariat intermediate (loop in the intron) - 1 exon detached
3. The exon’s free 3′-OH group displaces the 3′ end of the intron, forming a phosphodiester bond with the 5′-terminal phosphate of the other exon
= spliced product
How much energy does splicing involve?
Splicing process proceeds without free energy input
The transesterification reactions preserve the free energy of each cleaved phosphodiester bond through the formation of a new one
What is splicing mediated by?
Small nuclear RNAs (snRNAs) which form small nuclear ribonucleoproteins (snRNPs - snurps) in the spliceosome
The spliceosome carries out the two transesterification reactions
U1-U6 snRNAs are at the heart of spliceosome action - enhancing the nucleophilicity of the attacking OH group and stabilising the leaving group
There is a snRNP core protein, consisting of 7 Sm proteins - they bind to a conserved AAUUUGUGG sequence called Sm RNA motif
The Sm proteins form the heteroheptameric ring = 70 Å diameter
What is alternative mRNA splicing?
Alternative combinations of exons - yeilding multiple proteins from a single gene
= isoforms
Isoforms of genes lead to protein diversity
Exons can be retained or skipped, introns may be excised or retained, and the positions of 5′and 3′splice sites can be shifted to make exons longer or shorter
How does the spliceosome recognise small exons within the introns?
Regulatory proteins recognise these sequences and the splice site - to control which exons are spliced together
Exonic splicing enhancers (ESEs)
Exonic splicing silencers (ESSs)
Intronic splicing enhancers (ISEs)
Intronic splicing silencers (ISSs)
What can affect splicing?
15% of human genetic diseases are caused by point mutations - that result in pre-mRNA splicing defects
Activating preexisiting ‘cryptic splice sites’ or generating incorrect new ones
What happens to the mRNA after 5 cap, polyA tail and splicing?
mRNA can be edited - additional PTM using guide RNAs (gRNAs)
Mature eukaryotic mRNAs are actively transported from the nucleus to the cytoplasm
Mature mRNA is identified by m7G caps, polyA tails and the exon junction complex (EJC) that attach during splicing
Nucleoporins allow the diffusion of molecules but mRNA requires active transport to pass through a nuclear pore complex (NPC)
