Transcription and RNA Processing (1B) - Bio & Biochem

1. A mechanistic description focuses on 3' end processing of an mRNA encoding an insulin-responsive transporter. A point mutation disrupts the AAUAAA polyadenylation signal but leaves the coding region intact. In mutant cells, nuclear RNA accumulates as a longer transcript that extends beyond the normal 3' end, and cytosolic mRNA levels drop. Which outcome would be expected from this mutation?
   Answer: Reduced cleavage and polyadenylation lead to inefficient transcript termination and decreased mRNA export/stability, lowering cytosolic mRNA. This question addresses 3' end processing's role in mRNA maturation and stability. The polyadenylation signal enables cleavage and poly(D) addition, facilitating termination and export; mutation causes read-through and reduced cytosolic mRNA. The vignette describes longer nuclear RNA and dropped cytosolic levels due to the mutation. Choice D is correct as it links impaired processing to termination defects and instability. Choice C is wrong, assuming splicing compensation, misunderstanding 3' processing independence. To reason alike, compare nuclear versus cytosolic RNA upon 3' mutations. Check transcript lengths to detect read-through.
2. To probe how RNA processing influences metabolic adaptation, researchers inhibit the nuclear 5' capping enzyme complex for 60 minutes in proliferating cells. They observe that newly synthesized transcripts from a nucleotide biosynthesis gene accumulate in the nucleus, while cytosolic mRNA and protein levels fall. Total Pol II occupancy across the gene body remains similar to control. Which outcome would be expected if 5' capping is inhibited in this context?
   Answer: Reduced transcript stability and impaired nuclear export, decreasing cytosolic mature mRNA despite ongoing transcription. This question tests knowledge of 5' capping's role in mRNA stability, nuclear export, and translation. The 5' cap structure (7-methylguanosine) is added co-transcriptionally and is essential for mRNA stability, nuclear export via cap-binding proteins, and ribosome recruitment for translation. Inhibiting the capping enzyme produces uncapped transcripts that accumulate in the nucleus because they cannot be efficiently exported and are rapidly degraded. The correct answer B explains that uncapped mRNAs have reduced stability and impaired nuclear export, decreasing cytosolic mature mRNA levels. Option A incorrectly suggests enhanced translation of uncapped mRNA, but uncapped transcripts are actually poorly translated because ribosomes require the cap for efficient binding. Remember that 5' capping is one of the earliest RNA processing events and affects multiple downstream processes including stability, export, and translation.
3. A splicing regulator promotes skipping of exon 5 in a transcript encoding a citric acid cycle enzyme. Exon 5 length is 75 nucleotides and its exclusion does not shift the reading frame. Researchers inhibit the regulator and observe increased inclusion of exon 5 in mature mRNA. Which downstream outcome is most consistent with increased exon 5 inclusion, assuming protein expression occurs?
   Answer: A protein isoform containing an additional 25 amino acids relative to the exon-skipped isoform. This question tests understanding of alternative splicing consequences on protein structure, particularly how exon inclusion/exclusion affects the resulting protein isoform. When a 75-nucleotide exon (divisible by 3) is included rather than skipped, it adds 25 codons to the mRNA without disrupting the reading frame, resulting in a protein containing an additional 25 amino acids compared to the exon-skipped version. This demonstrates how alternative splicing generates protein diversity - different combinations of exons produce proteins with distinct sequences and potentially different functions. The maintenance of reading frame is crucial because 75 nucleotides equals exactly 25 codons, preventing frameshifts that would produce truncated proteins. Choice A incorrectly predicts a frameshift, but the question explicitly states the reading frame is maintained, while choice C wrongly suggests splicing doesn't affect coding sequences. When analyzing alternative splicing outcomes, calculate whether exon length is divisible by 3 to predict frame maintenance, and remember that in-frame exon inclusion adds amino acids proportional to exon length divided by 3.
4. An experimental vignette tests whether a metabolite (citrate) affects transcription of an acetyl-CoA utilization gene through a transcription factor (TF-Cit). TF-Cit binds DNA only when acetylated. Cells treated with citrate show increased TF-Cit acetylation and increased nascent RNA. When a lysine-to-arginine mutation prevents TF-Cit acetylation, citrate no longer increases nascent RNA. Which statement is most consistent with the mechanism of citrate action in this system?
   Answer: Citrate promotes TF-Cit acetylation, enabling DNA binding and transcriptional activation at the promoter. This question explores metabolite sensing in transcription regulation. Citrate induces TF-Cit acetylation for DNA binding and activation, increasing nascent RNA; mutation prevents this. The vignette links acetylation to RNA increase. Choice D is correct as it describes acetylation-enabled activation. Choice B fails by suggesting repression relief, but data show activation loss in mutant. To reason alike, test modifications and binding in regulation. Use mutants to confirm activation mechanisms.
5. Researchers compare transcription of a peroxisomal beta-oxidation enzyme gene in two conditions: high fatty acids vs low fatty acids. A ligand-activated nuclear receptor (NR) binds an enhancer upstream. When NR is activated, enhancer RNA (eRNA) production increases and promoter–enhancer looping (measured by 3C) increases. Mature mRNA increases. Which inference is most consistent with these observations?
   Answer: NR activation likely increases transcription initiation by promoting enhancer–promoter communication and recruitment of transcriptional machinery. This question tests enhancer-mediated transcriptional activation. NR activation increases eRNA and looping, facilitating initiation and mature mRNA increase. High fatty acids induce the changes via NR. Choice A is correct as it infers enhanced initiation through enhancer-promoter interactions. Choice D fails by suggesting decreased initiation, contradicting mRNA increase. For parallels, measure eRNA and looping for activation evidence. Compare conditions to infer regulatory mechanisms.
6. A research group is testing how coupling between transcription and RNA processing affects metabolic gene output in cultured human hepatocytes. Cells are treated with either vehicle or a small molecule that inhibits the CTD Ser2 phosphorylation of RNA polymerase II (Pol II) without directly inhibiting Pol II catalysis. After 2 hours, researchers quantify (i) total nascent RNA at the gene locus by intron-specific qPCR and (ii) mature cytosolic mRNA by exon–exon junction qPCR for a fatty-acid oxidation enzyme. Total nascent RNA changes minimally, but mature cytosolic mRNA decreases markedly. Based on this vignette, which conclusion is most consistent with RNA processing efficiency under Ser2 phosphorylation inhibition?
   Answer: Spliceosome recruitment to the nascent transcript is reduced, decreasing co-transcriptional splicing and lowering mature mRNA despite near-normal transcription initiation. This question tests understanding of how RNA polymerase II CTD phosphorylation couples transcription with RNA processing, specifically splicing. CTD Ser2 phosphorylation serves as a binding platform for splicing factors, facilitating co-transcriptional recruitment of the spliceosome to nascent transcripts. The scenario shows normal transcription initiation (unchanged nascent RNA) but reduced mature mRNA, indicating a processing defect rather than a transcription defect. The correct answer A explains that inhibiting Ser2 phosphorylation reduces spliceosome recruitment, decreasing splicing efficiency without affecting transcription initiation. Option C incorrectly suggests reduced promoter binding, which would decrease nascent RNA levels, contradicting the data. A key check for similar problems: if nascent RNA is normal but mature mRNA is reduced, the defect lies in RNA processing (splicing, capping, or polyadenylation), not in transcription initiation.
7. Researchers evaluate whether a small molecule (Mol-S) improves expression of a urea cycle enzyme by altering RNA processing. Mol-S does not change promoter occupancy by RNA polymerase II, but it increases the abundance of mature mRNA while decreasing unspliced pre-mRNA. Total RNA levels from the gene (pre-mRNA + mRNA) are unchanged. Based on the vignette, which conclusion is most consistent with Mol-S action?
   Answer: Mol-S likely enhances splicing efficiency or pre-mRNA processing, shifting RNA from unspliced to spliced forms without increasing transcription. This question probes small molecule effects on RNA processing versus transcription. Mol-S shifts unspliced to spliced mRNA without changing total RNA or promoter occupancy, indicating processing enhancement. The vignette shows unchanged total but altered ratios, consistent with splicing improvement. Choice C is correct as it infers enhanced processing efficiency. Choice B fails by linking to initiation despite unchanged occupancy, misunderstanding occupancy as rate proxy. In parallels, compare total and processed RNA levels. Use occupancy assays to distinguish transcription effects.
8. Researchers study alternative splicing of a mitochondrial fatty-acid oxidation regulator pre-mRNA containing exon 4, which introduces a premature stop codon when included. They mutate a short intronic splicing enhancer (ISE) downstream of exon 4 that normally binds a serine/arginine-rich (SR) protein. In nutrient-replete conditions, wild-type cells produce mostly exon 4–skipped mRNA; under low glucose, exon 4 inclusion increases. After ISE mutation, exon 4 inclusion remains high even in nutrient-replete media. Which outcome would be expected if the SR protein that binds the ISE is inhibited in wild-type cells?
   Answer: Exon 4 inclusion increases because loss of SR-mediated enhancer activity reduces exon definition and favors exon skipping. This question assesses knowledge of alternative splicing regulation by splicing factors in response to nutrient conditions. SR proteins typically bind exonic or intronic enhancers to promote exon inclusion by facilitating spliceosome assembly. Here, the ISE downstream of exon 4 binds an SR protein to repress inclusion under nutrient-replete conditions, but mutation or inhibition shifts to inclusion. Choice C is correct because inhibiting the SR protein mimics ISE mutation by reducing enhancer activity, weakening exon definition and favoring skipping, but the question expects increased inclusion due to loss of repression. Choice A fails as it misattributes SR proteins as general repressors, a common error confusing them with hnRNPs. For similar problems, verify if the factor is an enhancer or silencer by observing inclusion patterns upon loss. Also, consider nutrient context to predict shifts in splicing outcomes.
9. Researchers use an in vitro transcription system to study RNA polymerase II fidelity on a gene encoding a metabolic enzyme. They compare normal NTP concentrations to low ATP conditions. Under low ATP, short abortive transcripts accumulate near the promoter, while full-length transcripts decrease. Adding a general transcription factor that stabilizes the open complex partially rescues full-length transcription. Which explanation best fits these observations?
   Answer: Low ATP limits early transcript extension and promoter escape, increasing abortive initiation; stabilizing the open complex improves productive initiation. This question investigates nucleotide availability's impact on transcription initiation and elongation. Low ATP hinders promoter escape, causing abortive transcripts, while stabilizing factors aid productive initiation. The in vitro system shows abortive accumulation under low ATP, rescued by open complex stabilization. Choice A is correct as it explains low ATP limiting extension, with stabilization improving initiation. Choice B fails by linking to splicing, a misconception ignoring ATP's role in polymerase kinetics. For parallels, evaluate transcript lengths under NTP variations. Use rescue experiments to confirm initiation mechanisms.
10. A lab investigates why a gluconeogenic enzyme transcript is low in a mutant cell line despite normal promoter occupancy by RNA polymerase II. The gene contains an intron with a weak 3' splice site. The mutant lacks a functional U2AF65 protein (normally binds the polypyrimidine tract near the 3' splice site). Nuclear run-on assays show similar transcription rates between wild-type and mutant. However, RT-qPCR detects increased intron retention in the mutant. Which outcome would be expected if U2AF65 function is restored in the mutant cells?
    Answer: Intron retention decreases and mature mRNA increases without requiring a change in transcription rate. This question probes the coupling of splicing factors to transcription and mRNA maturation. U2AF65 aids spliceosome assembly by binding the 3' splice site, promoting intron removal for mature mRNA formation. The mutant shows intron retention despite normal transcription, indicating a splicing defect. Choice A is correct because restoring U2AF65 would enhance splicing, reducing retention and increasing mature mRNA without altering transcription rate. Choice B fails as it incorrectly assumes U2AF65 recruits polymerase, a misunderstanding of its role in splicing rather than initiation. In analogous cases, assess if defects affect processing efficiency versus transcription by comparing run-on assays and spliced fractions. Use RT-qPCR for introns to detect retention patterns.
11. A lab studies regulation of glycolytic flux by transcriptional control of a rate-limiting enzyme. Under low-glucose conditions, a DNA-binding transcription factor (TF-L) is phosphorylated and binds an upstream enhancer. ChIP-qPCR confirms increased TF-L occupancy, and nuclear run-on assays show increased RNA synthesis at the target gene. However, cytosolic mature mRNA rises only modestly. The team suspects that TF-L primarily enhances transcription initiation but does not improve RNA processing. Which statement best describes the role of TF-L that is most consistent with the data?
    Answer: TF-L promotes transcription initiation by facilitating assembly of the Pol II preinitiation complex, while RNA processing remains rate-limiting downstream. This question tests understanding of how transcription factors can differentially affect transcription initiation versus RNA processing efficiency. TF-L binding to the enhancer increases RNA synthesis (shown by nuclear run-on assays) but produces only modest increases in mature mRNA, suggesting a bottleneck in RNA processing. The correct answer B explains that TF-L promotes transcription initiation by facilitating preinitiation complex assembly, but doesn't improve downstream RNA processing steps like splicing or 3' end formation. Option D incorrectly suggests decreased elongation speed would reduce run-on signal, contradicting the observed increase in RNA synthesis. A critical principle: transcription factors that enhance initiation don't necessarily improve RNA processing efficiency, so increased transcription doesn't always yield proportional increases in mature mRNA. When interpreting such data, compare changes in nascent RNA synthesis with changes in mature mRNA to identify rate-limiting steps.
12. To study transcription fidelity in a metabolic stress response, scientists introduce a point mutation into the active site of RNA polymerase II that increases misincorporation frequency but does not alter promoter recognition. Cells show similar levels of pre-mRNA production from a stress-response enzyme gene, but the mature mRNA is degraded more rapidly, and protein levels fall. Which mechanism best explains the reduced mature mRNA abundance?
    Answer: Misincorporations in the transcript increase the likelihood of aberrant splice site creation or premature stop codons, promoting nuclear quality control and decay. This question tests understanding of transcription fidelity and nuclear RNA quality control mechanisms. RNA polymerase II normally has high fidelity, but mutations that increase misincorporation produce error-containing transcripts that may create cryptic splice sites or premature termination codons (PTCs). Nuclear quality control mechanisms detect and degrade aberrant transcripts before export. The correct answer B explains that misincorporations can create aberrant splice sites or PTCs, triggering nuclear decay pathways. Option A incorrectly places the problem at translation (amino acid incorporation), but the question specifically addresses transcription fidelity affecting RNA sequence. Remember that cells have multiple RNA quality control checkpoints, including nuclear surveillance that prevents export of aberrant transcripts, protecting the cell from producing dysfunctional proteins.
13. Investigators engineer a minigene encoding a mitochondrial enzyme involved in the TCA cycle. The pre-mRNA contains one intron with a canonical 5' splice site but a mutated 3' splice site polypyrimidine tract. Nuclear RNA sequencing shows increased intron retention and reduced exon–exon junction reads, while total transcription at the locus (measured by Pol II occupancy) is unchanged. Which outcome would be expected if the 3' splice site recognition factor U2AF is further inhibited in these cells?
    Answer: Further reduction in mature mRNA due to impaired recruitment of U2 snRNP to the branch point, increasing intron retention. This question tests knowledge of splice site recognition and the role of U2AF in 3' splice site selection during pre-mRNA splicing. U2AF (U2 auxiliary factor) recognizes the polypyrimidine tract at the 3' splice site and recruits U2 snRNP to the branch point, essential steps in spliceosome assembly. The minigene already has a mutated polypyrimidine tract causing intron retention, and further inhibiting U2AF would exacerbate this splicing defect. The correct answer B predicts further reduction in mature mRNA due to even more impaired U2 snRNP recruitment. Option A incorrectly suggests U2AF inhibition would improve 5' splice site recognition, but U2AF specifically acts at the 3' splice site, not the 5' site. Remember that splicing requires recognition of both 5' and 3' splice sites, and defects in either will increase intron retention and decrease exon-exon junction formation.
14. A lab studies a transcription factor (TF-Y) that binds a response element in the promoter of a lipid uptake gene. TF-Y also recruits a coactivator with histone acetyltransferase (HAT) activity. When TF-Y is overexpressed, promoter-proximal nucleosomes show increased acetylation and nascent RNA increases. A TF-Y mutant that still binds DNA but cannot bind the coactivator fails to increase nascent RNA. Which statement best describes the role of the coactivator in TF-Y–dependent gene expression?
    Answer: The coactivator increases transcription by acetylating histones to promote chromatin accessibility and RNA polymerase II recruitment. This question evaluates coactivator functions in transcription activation. The HAT coactivator acetylates histones, enhancing chromatin accessibility and polymerase recruitment via TF-Y. Overexpression increases acetylation and nascent RNA, but the mutant fails, highlighting coactivator necessity. Choice A is correct as it describes the coactivator's role in activation through acetylation. Choice B fails by attributing to mRNA methylation, confusing with other modifications. In similar cases, use ChIP for acetylation and nascent RNA assays. Test mutants to confirm coactivator dependencies.
15. Researchers analyze an alternatively spliced transcript encoding an enzyme in amino acid catabolism. Under fasting conditions, cells show increased inclusion of exon 4, producing a protein isoform with higher catalytic activity. A splicing regulator (SR protein) is found to bind an exonic splicing enhancer (ESE) within exon 4. When the ESE is mutated, exon 4 inclusion drops even though transcription rate is unchanged. Which outcome would be expected if the SR protein is inhibited while the ESE remains intact?
    Answer: Exon 4 inclusion decreases because SR-dependent enhancer binding promotes recruitment of splicing machinery to favor exon definition. This question tests understanding of SR proteins and exonic splicing enhancers (ESEs) in alternative splicing regulation. SR proteins are serine/arginine-rich proteins that bind ESEs and recruit splicing machinery to promote exon inclusion through exon definition. The scenario shows that SR protein binding to the ESE in exon 4 promotes its inclusion, as ESE mutation reduces inclusion. The correct answer B predicts that SR protein inhibition would decrease exon 4 inclusion because SR proteins are needed to recruit splicing factors for exon definition. Option A incorrectly suggests SR proteins repress splicing, but they actually promote exon inclusion when bound to ESEs. Remember that alternative splicing is regulated by the balance of enhancers (ESEs/ISEs) and silencers (ESSs/ISSs), with SR proteins typically promoting inclusion via enhancer sequences.
16. In a hepatocyte-derived cell line, investigators test whether a nutrient-sensitive transcription factor (TF-X) coordinates expression of a glycolytic enzyme gene. Cells are transfected with a reporter plasmid in which luciferase is driven by the enzyme's promoter containing a TF-X binding site. TF-X is activated by dephosphorylation during high glucose. Four conditions are compared for 6 hours: control, high glucose, high glucose + TF-X siRNA, and high glucose + an inhibitor of spliceosome assembly (blocks U1 snRNP recruitment). Luciferase activity and the fraction of spliced luciferase mRNA (measured by RT-qPCR across the exon–exon junction) are recorded. Which conclusion is most consistent with the data pattern expected if TF-X primarily increases transcription initiation rather than RNA processing efficiency?
    Answer: High glucose increases luciferase activity without changing the spliced mRNA fraction, and TF-X siRNA abolishes the activity increase while spliceosome inhibition lowers the spliced fraction but does not mimic TF-X siRNA. This question tests understanding of transcription initiation versus RNA processing in gene expression regulation. Transcription initiation involves recruitment of RNA polymerase II to the promoter, while RNA processing includes splicing of pre-mRNA to form mature mRNA. In this scenario, TF-X is activated by high glucose and binds the promoter, with experiments comparing luciferase activity and spliced mRNA fractions under various conditions. Choice D is correct because if TF-X primarily enhances initiation, high glucose increases activity without altering splicing efficiency, siRNA blocks the initiation boost, and spliceosome inhibition affects splicing but not the initiation-driven activity. A common misunderstanding is that transcription factors directly affect splicing, as in choice B, but here siRNA does not reverse splicing changes since TF-X acts at initiation. To apply this reasoning elsewhere, check if perturbations affect total transcript levels versus processed fractions. Additionally, distinguish initiation effects by seeing if nascent RNA or activity changes independently of processing inhibitors.
17. A team uses an in vitro transcription system with purified RNA polymerase II and general transcription factors to transcribe a DNA template encoding a stress-response enzyme. They add either (i) all four rNTPs, (ii) all four dNTPs, or (iii) all four rNTPs plus an RNase that degrades single-stranded RNA. Which condition is expected to yield the highest amount of full-length RNA transcript?
    Answer: All four rNTPs, because RNA polymerase II requires ribonucleotides and the transcript is not degraded during synthesis. This question tests fundamental knowledge of RNA polymerase II substrate requirements and the chemical differences between RNA and DNA synthesis. RNA polymerase II exclusively uses ribonucleoside triphosphates (rNTPs) as substrates to synthesize RNA, incorporating the 2'-OH group that distinguishes RNA from DNA, and cannot utilize deoxyribonucleoside triphosphates (dNTPs) which lack this hydroxyl group. In an in vitro transcription system with all necessary components, providing all four rNTPs allows continuous RNA synthesis without degradation, yielding the maximum amount of full-length transcript. The presence of general transcription factors ensures proper initiation and elongation. Choice A incorrectly suggests Pol II can use dNTPs, which would be like asking a chef to bake bread with metal instead of flour - the enzyme's active site specifically requires the 2'-OH for catalysis. Adding RNase (choice C) would degrade the RNA product as it's synthesized, reducing yield rather than increasing it. Remember that RNA polymerases are absolutely specific for rNTPs due to active site geometry and catalytic mechanism requirements.
18. Investigators examine an mRNA encoding a rate-limiting enzyme in heme synthesis. A mutant cell line has normal transcription initiation and normal splicing, but produces mRNAs with heterogeneous (variable) 3' ends and reduced steady-state mRNA levels. Sequencing indicates frequent use of cryptic polyadenylation sites within the last exon. Which defect is most likely to produce this pattern?
    Answer: Impaired recognition of the canonical polyadenylation signal, increasing usage of alternative cleavage/polyadenylation sites and destabilizing the mRNA. This question tests understanding of 3' end formation and the role of polyadenylation signals in mRNA stability. The normal transcription and splicing with heterogeneous 3' ends points to a defect in recognition of the canonical polyadenylation signal (typically AAUAAA). When this signal is not properly recognized by cleavage and polyadenylation factors, the machinery uses weaker, cryptic polyadenylation sites within the last exon, producing variable 3' ends. These alternatively processed mRNAs often have shorter 3' UTRs and may lack important stability elements, leading to reduced steady-state levels through increased degradation. The defect is specific to 3' end processing rather than transcription or splicing, as these processes occur normally. Students might incorrectly focus on splicing defects when seeing mRNA heterogeneity, but the presence of normal splicing with variable 3' ends specifically indicates polyadenylation problems. A key diagnostic principle: when transcription and splicing are normal but mRNAs have variable 3' ends with reduced stability, the defect involves polyadenylation signal recognition and alternative site usage.
19. A group measures transcription efficiency of a mitochondrial biogenesis regulator using an in vitro Pol II transcription system supplemented with nuclear extract. When ATP is depleted (while NTPs for RNA synthesis are maintained), the amount of full-length RNA product decreases, and short abortive transcripts accumulate. Splicing is not assayed in this system. Based on the vignette, which conclusion is most consistent with the effect of ATP depletion on transcription output?
    Answer: ATP depletion primarily impairs helicase/kinase activities required for promoter clearance and early elongation, increasing abortive initiation products. This question tests understanding of ATP requirements in transcription beyond providing substrates for RNA synthesis. ATP is required for multiple steps including promoter clearance, where helicases unwind DNA, and CTD kinases phosphorylate Pol II for the transition from initiation to elongation. ATP depletion causes accumulation of short abortive transcripts because Pol II cannot efficiently clear the promoter and enter productive elongation. The correct answer A explains that ATP powers helicases and kinases needed for promoter clearance and early elongation, and its depletion increases abortive initiation. Option B incorrectly invokes ribosome competition, but this is an in vitro transcription system without translation. A critical concept: transcription requires ATP not just as a substrate (ATP→AMP in RNA) but as an energy source for enzymes that facilitate Pol II progression through different transcription stages.
20. Researchers test whether a transcription factor (TF-M) that responds to amino acid availability activates transcription of an amino acid transporter gene by recruiting Mediator. In cells expressing TF-M, ChIP shows increased Mediator occupancy at the promoter and increased RNA polymerase II at the transcription start site. A TF-M mutant that binds DNA but cannot interact with Mediator shows normal DNA binding but low transcription. Which statement best describes TF-M's role?
    Answer: TF-M activates transcription by facilitating assembly of the preinitiation complex via Mediator recruitment, increasing initiation frequency. This question evaluates transcription factor mechanisms via Mediator. TF-M recruits Mediator to assemble the preinitiation complex, increasing initiation; mutant fails despite DNA binding. ChIP shows increased occupancy with wild-type TF-M. Choice A is correct as it describes Mediator-dependent activation. Choice B is wrong, attributing to splicing, confusing with post-transcriptional roles. For application, use ChIP and mutants to dissect recruitment. Monitor polymerase occupancy for initiation effects.

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