ROJW - basal transcription machinery Flashcards
Bacterial RNA Polymerase structure consist of:
2 ⍺ subunits, 1 β and β’ subunit, and a ω subunit
Eukaryotic RNA polymerase:
3 different types of Eukaryotic RNA pol responsible for transcribing different families of RNA
* RNA Pol I transcribes 5.7S, 18S and 28S rRNAs
o Located in the nucleolus and responsible for the formation of the nucleolus
* RNA Pol II transcribes all mRNAs
o Located in the nucleus
* RNA Pol III transcribes 5S rRNA, all tRNAs and other small RNAs
o Located in the nucleus
RNA polymerases can also be found in cellular components ex. Mitochondria and chloroplast which have their own genome
Eukaryotic RNA polymerase structure
- 12 subunits
- Some components’ sequence and tertiary structure are similar to bacterial RNAP
Comparison of RNA pol 2 structures to Bacterial RNA pol (similarities)
o B = RNA pol 2 (A,B,C = RNA pol 1, 2, 3)
o Numbers = according to molecular size of the subunits
Similarities:
* The 2 main subunits: RPB1 and RPB2 are similar to bacterial β’ and β subunits
* RPB3, RPB10, RPB11 and RPB12 are similar to the ⍺2 homodimer
* RPB6 is similar to ω
* Both form claw-like structures
* Both have magnesium ion coordinated by acidic residues in the active site where RNA synthesis occurs – Mg2+ is required as a cofactor for the addition/ polymerization of nucleotide
Comparison of RNA pol 2 structures to Bacterial RNA pol (differences)
Differences:
* Eukaryotic RNA pol has more subunits = additional complexity
o Ex. RPB7 & RPB4 which form stalk-like extensions
Bacterial RNA pol holoenzyme contains a sigma subunit that allows for recognition of specific promotors at -10 & -30 region without the help of any other transcription factors
Although eukaryotic RNAPs have a larger number of subunits, they do not have any obvious sequence-specific binding activities by themselves. They require the assistance of additional transcription factors. The additional factors are also complicated complexes in their own way (not just single polypeptide)
Basal transcription factors
Basal transcription factors that give low, basal levels of unregulated transcription RNA pol 2 basal machinery is a stepwise process
- TFIIA, TFIIB, TFIID recognize promoter elements
- TFIIF recruits RNA polymerase
- TFIIE, TFIIH bind downstream of polymerase and allow for the formation of the transcription bubble around the transcript start position, completing the basal transcription initiation complex
Gene-specific transcription factors
Gene-specific transcription factors that programs the basal transcription inhiation complex to give regulated levels of gene expression. (Regulate imitation properties of RNA polymerase)
* Proximal Gene-Specific Transcription Factors (<1kb away)
* Distal Gene-Specific Transcription Factors - involve interactions over large distances
But all have to physically interact, either directly or via mediator proteins, with the basal transcription machinery. This is possible due to the flexibility of DNA which can form loops.
Steps of basal transcription initiation complex formation:
- TFIID specifically recognize TATA box
o TATA box: 1st eukaryotic promotor element discovered
o At-rich sequence embedded in GC rich neighboring sequences
o Region: around -30 to -25 upstream of start site - TFIIA,TFIIB bind underneath on both sides of TBP-DNA complex to stabilize TFIID binding
- TFIIF binds to RNA pol 2 & facilitates delivery of the RNA pol 2 to the TFIID-TFIIB-DNA complex on the promoter
o Kink formed by TFIID binding to DNA is well-suited to fit into the RNA pol structure to form a compact complex where the promotor is bound by RNA pol and other basal transcription factors associate with RNA pol at various points - TFIIE, TFIIH bind downstream of polymerase and are responsible for 3 critical functions in transcription after the recruitment stage
* phosphorylation of RNA polymerase II (makes RNA pol 2 elongation-competent)
* promoter melting via a DNA helicase mechanism (opens up dsDNA to expose the template strands to RNA polymerase activity – forms transcription bubble)
* promoter clearance (allows for RNA polymerase to leave the initiation complex and start transcribing the gene)
What is TFIID
- TFIID is a multiprotein complex consisting of TATA-Binding Protein (TBP) and around a series of other subunits (TBP-Associated Factors; TAFs)
o Only TBP can bind in a sequence-specific manner to the TATA-box on its own
TBP structures
Has a satellite structure consisting of a beta sheet with alpha helices on top
Binding of TBP to TATA box on DNA induce kink formation
Since DNA is strongly kinked (depends on type of TATA box) at ~90°, binding of TBP to DNA itself is energetically inefficient
TATA box flanking sequences
- The TATA box flanking sequences (upstream & downstream of TATA box – but predominantly upstream) can form the BRE or TFIIB recognition element which allow for sequence specific contact of TFIID with DNA. Therefore, optimized TATA box sequences AND region flanking TATA box can result in stronger DNA binding because it allows for strong binding of both TBP and TFIID to DNA instead of only the TBP-DNA complex
o Viruses usually have optimal TATA box promotors because when they infect cells, they have to compete with the cell promotors
Experimental means of Detecting Transcription Factor Complexes binding to DNA:
Performing Electrophoretic Mobility Shift Assay (EMSA)
* Allow observation of the complex in a stepwise manner
* Promotor region of DNA is fluorescently tagged
o Lane 1: without any TF added, the promotor is of small size so will all run to the bottom of the gel, resulting in 1 dark band
o Lane 2: TBP added, forming TBP-DNA complex which runs slower on the gel – but only a small portion is bound to TBP– most DNA is still in free form, so see faint band in addition to dark band of DNA at the bottom of the gel
Binding of TBP to DNA itself is energetically inefficient because DNA = strongly bent
o Lane 3: TFIIB added – result in another band of larger Mw due to TBP-TFIIB-DNA complex. More intense band than free DNA – indicate that more DNA is involved in the complex formation, indicating that TFIIB indeed stabilizes TBP binding to DNA
Other bands = conformational heterogeneity
o Lane 4-7: RNA pol 2 titrated in from low to high conc. – result in another band of larger Mw indication RNAP-TFIIB-TBP-DNA complex which become clearer as higher conc. of RNA pol is added
Indicate that all of RNA pol added is able to bind DNA due to the presence of TFIIB-stabilized TBP binding to DNA (require the step of TFIIB-TBP-DNA complex formation prior to RNA pol binding
Other promotor proximal elements:
- Not all RNAPII-transcribed genes contain TATA-boxes (only 20-25% of human genome contains TATA boxes) – majority of human genes are transcribed from promotors that do not contain TATA box
- There are at least two further consensus motifs (other promotor proximal elements) that may be present:
o the ‘Initiator Element’ (‘IE’): sits around transcription start site (+1)
o the ‘Downstream Promoter Element’ (‘DPE’): located at the downstream end of the promotor, within the transcribed region after transcription start site (+1) - (more constant in position than TATA box)
o others = continuously being discovered - these other promotor-proximal regions are recognized by TBP-associated factors (TAFs) which are the other components of the TFIID complex
- different genes use different combinations of promoter promoter-proximal elements
Phosphorylation of RNA pol 2
The largest subunit of RNA pol 2 (RPB1) has a long C-terminal domain consisting of several repeats of a 7-residues sequence (consensus: YSPTSPS)
* 26 repeats in yeast, 52 repeats in humans
The sequence is unusual in the sense that it contains 2 proline positions which is an amino acid that is incompatible with secondary structure formation. This indicates that the C-terminal domain of RNA pol 2 does not form complex 3D structure, but act more as a C-terminal tail.
* Since the C-terminal tail does not take up 3D space, it does not diffract Xray signals so is not observed in Xray structure of RNA pol 2
The sequence also has serine and threonine residues which has OH groups that allow them to be post-translationally modified
Therefore, this CTD tail is phosphorylated by TFIIH kinase
* Hypophosphorylated (‘low levels of phosphorylation’) CTD is associated with the initiation complex
* Upon binding of TFIIH, the CTD tail is phosphorylated
* Hyperphosphorylated (‘extensively phosphorylated’) CTD turns RNA pol 2 to an elongation-competent form
* The phosphorylation of CTD domain also attracts a number of enzymes that bind to the CTD & travel along with RNA pol during elongation to carry out enzymatic functions. They regulate a number of other processes associated with transcription:
o 5’ capping
o assembly of spliceosomes
o binding of the cleavage/polyadenylation complex (allowing stalling of transcription & formation of polyA tail)
Promotor melting
- TFIIH is required for ‘melting’ the dsDNA around the transcription start site to form the transcription bubble, providing the RNA pol with ssDNA template for transcription
- Transcript initiation can only occur if the transcription bubble is present
- BUT Transcription bubble is formed in a transient manner
o The melted promoter is intrinsically unstable with a measured half-life ~ 45 seconds (vs stable bacterial open complex)
o If transcription initiation does not occur within this time span, the melted promoter will revert back to the dsDNA configuration. The transcription bubble can only be re-formed and sustained by TFIIH helicase activity which requires continued ATP-hydrolysis
o Therefore, this is an important control point for regulating the rate of initiation. By regulating TF2H helicase activity, it can influence the transcription initiation rate of RNA pol2
