Which RNA polymerase is required for transcription of mRNA in eukaryotes?
This conclusion is solely based on deletion (anchor-away) of only TAF1, a specific component of TFIID. Inclusion of two other TFIID-specific TAFs would have significant impact on this conclusion. Show Analyzing additional TAFs by anchor-away has minimal value given the point of the paper. Many TAF-inactivation experiments indicating that TFIID-specific TAFs selectively affect transcription. Regarding TAFs, the major point of our paper is to invalidate the opposite claim of Warfield et al., 2017. We do this by measuring TAF:GTF occupancies upon depletion of TAF1 (not done previously for any TAF-depletion experiment) and by re-analyzing the Warfield et al. genome-wide experiment (i.e. by restricting the analysis to genes whose expression is above the background). We also emphasize and discuss an interesting difference (discovered but largely ignored by Warfield et al.) with respect to the importance of TFIID in YPD vs. SC medium. Nevertheless, while not doing any new experiments ourselves, we did address this comment by analyzing Warfield et al. data on two other TFIID-specific TAFs (TAF11 and TAF13). The results (Figure 7—figure supplement 2) clearly demonstrate that 3 different TFIID-specific TAFs behave selectively and similarly with respect to the TFIID-SAGA distinction.
The suggestion of inhibiting elongation sounds good, but there is no known factor or drug that specifically inhibits elongation in yeast cells. More importantly, the point of the uracil experiment is to recreate the classic biochemical definition of the PIC (i.e. absence of NTPs) in vivoand assess its stability in vivo. There is no criticism of the experiment or the conclusions that arise. However, to address this suggestion, we now include an experiment done long ago in which we analyzed PIC and transcription levels in FACT-depleted cells. FACT is a histone chaperone that travels with elongating Pol II, is important for elongation through chromatin templates in vitro, and does not associate with promoters. Interestingly, as shown in new Figure 6, FACT depletion strongly reduces Pol II transcription genome-wide (already published by Pathak et al., 2018), and TFIIB occupancy at promoters. We recognize that the molecular basis of this observation is unknown, but we do think it is an interesting connection between elongation and PIC formation/stability worth reporting. We are not willing to do more experiments on FACT, so if our current Figure 6 is not acceptable to the reviewers, we will simply remove it. As mentioned above, the FACT experiment is not directly relevant for the uracil-depletion experiment, which is a key conclusion of the paper.
The suggestion to look at other GTFs upon depletion of an individual GTF is unnecessary and would take a vast amount of work. First of all, our experiments already involve 2 GTFs, TBP and Pol II. Second, numerous biochemical experiments and high-resolution structural knowledge make it virtually impossible that any GTF or combination of GTFs can stably interact with the promoter in the absence of TBP. Moreover, to do this suggestion properly, we would have to do a vast number of genome-wide experiments in which we look at every GTF for every individual GTF depletion. Otherwise, one could always say that upon depletion of factor X, factor Y remains associated with a subset of promoters.
The reviewer correctly notes that previous results that TBP can remain at TAF-dependent promoters upon thermal inactivation of TFIIB or Mediator (Med17 subunit) are in apparent conflict with our conclusion that partial PICs do not exist at appreciable levels in vivo. There are several possible explanations, not mutually exclusive, for this apparent discrepancy. First, the ts mutants used in the earlier experiments may not (and are unlikely to) completely inactivate TFIIB or Mediator. Because these experiments were done so long ago, ChIP was not done to assess the promoter occupancy of these factors upon thermal inactivation, and the transcriptional results relied on mRNA levels that are subject to mRNA stability issues. Furthermore, as these mRNA measurements were made only 45 minutes after the ts shift, considerable mRNA remained from before the shift (i.e. wasn’t degraded), thereby making it impossible to measure low/modest levels of transcription. In this regard, our more recent experiments using Pol II occupancy as an assay indicate that substantial transcription does occur in the Med17 ts mutant (and anchor-away allele), and transcription and TBP occupancy is more efficient at TAF-dependent promoters (Petrenko et al., 2017). Second, as the measurements in our current paper are made 1 hr after GTF depletion, it is possible that partial PICs might be metastable and exist at earlier time points (not so easy to distinguish true partial PICs from incomplete depletion). Thus, our conclusion that partial PICs do not exist at appreciable levels is correct. However, we now discuss these issues in the revised manuscript.
We now mention the activator-specificity of TFIID recruitment to ribosomal protein promoters and cite the relevant papers. We note that this fact is largely irrelevant to the conclusions of our paper, which are concerned with whether TAFs are generally required or selectively important for transcription.
We have cited the papers mentioned in this comment. We note that these comments are related to the role of NuA4 at ribosomal protein promoters and the role of SAGA, subjects that are only peripherally related to the experiments and conclusions here.
The difference between Pol II occupancy measurements at +200- to +400 vs. those at 0 to +100 is very subtle. In both cases, the Warfield et al. analysis of “all genes” shows very little difference between TATA-containing and TATA-less promoters, whereas our analysis of the 500 most well-expressed genes (i.e. the ones where there is reliable data) shows considerable difference. We do not know why the measurements at these two different regions are subtly different and hence haven’t commented on this in the revised paper. Pol II pausing is not relevant here, because numerous genome-wide experiments indicate that pausing does not occur in yeast, unlike what occurs in many other eukaryotic organisms. Which RNA polymerase transcribes mRNA in eukaryotes?All eukaryotes have three different RNA polymerases (RNAPs) which transcribe different types of genes. RNA polymerase I transcribes rRNA genes, RNA polymerase II transcribes mRNA, miRNA, snRNA, and snoRNA genes, and RNA polymerase III transcribes tRNA and 5S rRNA genes.
Is RNA polymerase 1 used in transcription?RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA).
Is RNA polymerase required for eukaryotic transcription?Like prokaryotic cells, the transcription of genes in eukaryotes requires the action of an RNA polymerase to bind to a DNA sequence upstream of a gene in order to initiate transcription.
What is RNA polymerase 1 vs 3?RNA Polymerase I is an enzyme that transcribes ribosomal RNAs. RNA Polymerase II is an enzyme that transcribes precursors of mRNAs. RNA Polymerase III is an enzyme that transcribes tRNAs. It transcribes all rRNAs except the 5S rRNA component.
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