Publications by authors named "Craig D Kaplan"

43 Publications

Germline mutation in : a heterogeneous, multi-systemic developmental disorder characterized by transcriptional dysregulation.

HGG Adv 2021 Jan 20;2(1). Epub 2020 Nov 20.

Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA.

germline variation in was recently reported to associate with a neurodevelopmental disorder. We report twelve individuals harboring putatively pathogenic or inherited variants in , detail their phenotypes, and map all known variants to the domain structure of and crystal structure of RNA polymerase II. Affected individuals were ascertained from a local data lake, pediatric genetics clinic, and an online community of families of affected individuals. These include six affected by missense variants (including one previously reported individual), four clinical laboratory samples affected by missense variation with unknown inheritance-with yeast functional assays further supporting altered function-one affected by a in-frame deletion, and one affected by a C-terminal frameshift variant inherited from a largely asymptomatic mother. Recurrently observed phenotypes include ataxia, joint hypermobility, short stature, skin abnormalities, congenital cardiac abnormalities, immune system abnormalities, hip dysplasia, and short Achilles tendons. We report a significantly higher occurrence of epilepsy (8/12, 66.7%) than previously reported (3/15, 20%) (p value = 0.014196; chi-square test) and a lower occurrence of hypotonia (8/12, 66.7%) than previously reported (14/15, 93.3%) (p value = 0.076309). -related developmental disorders likely represent a spectrum of related, multi-systemic developmental disorders, driven by distinct mechanisms, converging at a single locus.
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http://dx.doi.org/10.1016/j.xhgg.2020.100014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928427PMC
January 2021

A genome wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity.

J Biol Chem 2021 Mar 1:100485. Epub 2021 Mar 1.

Department of Biochemistry and Biophysics, MS 3474, Texas A&M University, College Station, TX 77843, USA. Electronic address:

Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or by supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.
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http://dx.doi.org/10.1016/j.jbc.2021.100485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027276PMC
March 2021

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler.

Bioessays 2020 07 3;42(7):e2000002. Epub 2020 Jun 3.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15260, USA.

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.
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http://dx.doi.org/10.1002/bies.202000002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329613PMC
July 2020

Universal promoter scanning by Pol II during transcription initiation in Saccharomyces cerevisiae.

Genome Biol 2020 06 2;21(1):132. Epub 2020 Jun 2.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15260, USA.

Background: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function.

Results: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model.

Conclusions: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.
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http://dx.doi.org/10.1186/s13059-020-02040-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265651PMC
June 2020

Organismal benefits of transcription speed control at gene boundaries.

EMBO Rep 2020 04 27;21(4):e49315. Epub 2020 Feb 27.

Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Frederiksberg, Denmark.

RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto-immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read-through transcription. The accelerated transcription mutant NRPB2-Y732F exhibits increased association with 5' splice site (5'SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.
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http://dx.doi.org/10.15252/embr.201949315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132196PMC
April 2020

High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier.

Elife 2019 07 31;8. Epub 2019 Jul 31.

Institute for Quantitative Biosciences-QB3, University of California, Berkeley, Berkeley, United States.

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.
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http://dx.doi.org/10.7554/eLife.48281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744274PMC
July 2019

Functional assays for transcription mechanisms in high-throughput.

Methods 2019 04 20;159-160:115-123. Epub 2019 Feb 20.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address:

Dramatic increases in the scale of programmed synthesis of nucleic acid libraries coupled with deep sequencing have powered advances in understanding nucleic acid and protein biology. Biological systems centering on nucleic acids or encoded proteins greatly benefit from such high-throughput studies, given that large DNA variant pools can be synthesized and DNA, or RNA products of transcription, can be easily analyzed by deep sequencing. Here we review the scope of various high-throughput functional assays for studies of nucleic acids and proteins in general, followed by discussion of how these types of study have yielded insights into the RNA Polymerase II (Pol II) active site as an example. We discuss methodological considerations in the design and execution of these experiments that should be valuable to studies in any system.
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http://dx.doi.org/10.1016/j.ymeth.2019.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589137PMC
April 2019

Perturbations of Transcription and Gene Expression-Associated Processes Alter Distribution of Cell Size Values in .

G3 (Bethesda) 2019 01 9;9(1):239-250. Epub 2019 Jan 9.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843

The question of what determines whether cells are big or small has been the focus of many studies because it is thought that such determinants underpin the coupling of cell growth with cell division. In contrast, what determines the overall pattern of how cell size is distributed within a population of wild type or mutant cells has received little attention. Knowing how cell size varies around a characteristic pattern could shed light on the processes that generate such a pattern and provide a criterion to identify its genetic basis. Here, we show that cell size values of wild type cells fit a gamma distribution, in haploid and diploid cells, and under different growth conditions. To identify genes that influence this pattern, we analyzed the cell size distributions of all single-gene deletion strains in We found that yeast strains which deviate the most from the gamma distribution are enriched for those lacking gene products functioning in gene expression, especially those in transcription or transcription-linked processes. We also show that cell size is increased in mutants carrying altered activity substitutions in Rpo21p/Rpb1, the largest subunit of RNA polymerase II (Pol II). Lastly, the size distribution of cells carrying extreme altered activity Pol II substitutions deviated from the expected gamma distribution. Our results are consistent with the idea that genetic defects in widely acting transcription factors or Pol II itself compromise both cell size homeostasis and how the size of individual cells is distributed in a population.
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http://dx.doi.org/10.1534/g3.118.200854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325893PMC
January 2019

Wide-ranging and unexpected consequences of altered Pol II catalytic activity in vivo.

Nucleic Acids Res 2017 05;45(8):4431-4451

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.

Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin immunoprecipitation can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants' growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.
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http://dx.doi.org/10.1093/nar/gkx037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416818PMC
May 2017

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.

PLoS Genet 2016 Nov 29;12(11):e1006321. Epub 2016 Nov 29.

Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas.

The active sites of multisubunit RNA polymerases have a "trigger loop" (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.
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http://dx.doi.org/10.1371/journal.pgen.1006321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127505PMC
November 2016

Pairs of promoter pairs in a web of transcription.

Authors:
Craig D Kaplan

Nat Genet 2016 08;48(9):975-6

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.

A new analysis has characterized a fundamental building block of complex transcribed loci. Constellations of core promoters can generally be reduced to pairs of divergent transcription units, where the distance between the pairs of transcription units correlates with constraints on genomic context, which in turn contribute to transcript fate.
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http://dx.doi.org/10.1038/ng.3649DOI Listing
August 2016

The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA.

Nature 2016 07 6;535(7612):444-7. Epub 2016 Jul 6.

The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.
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http://dx.doi.org/10.1038/nature18622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961592PMC
July 2016

Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae.

G3 (Bethesda) 2016 08 9;6(8):2489-504. Epub 2016 Aug 9.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843

The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants.
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http://dx.doi.org/10.1534/g3.116.030346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978902PMC
August 2016

RNA Polymerase II Trigger Loop Mobility: INDIRECT EFFECTS OF Rpb9.

J Biol Chem 2016 Jul 18;291(28):14883-95. Epub 2016 May 18.

From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128

Rpb9 is a conserved RNA polymerase II (pol II) subunit, the absence of which confers alterations to pol II enzymatic properties and transcription fidelity. It has been suggested previously that Rpb9 affects mobility of the trigger loop (TL), a structural element of Rpb1 that moves in and out of the active site with each elongation cycle. However, a biochemical mechanism for this effect has not been defined. We find that the mushroom toxin α-amanitin, which inhibits TL mobility, suppresses the effect of Rpb9 on NTP misincorporation, consistent with a role for Rpb9 in this process. Furthermore, we have identified missense alleles of RPB9 in yeast that suppress the severe growth defect caused by rpb1-G730D, a substitution within Rpb1 α-helix 21 (α21). These alleles suggest a model in which Rpb9 indirectly affects TL mobility by anchoring the position of α21, with which the TL directly interacts during opening and closing. Amino acid substitutions in Rpb9 or Rpb1 that disrupt proposed anchoring interactions resulted in phenotypes shared by rpb9Δ strains, including increased elongation rate in vitro Combinations of rpb9Δ with the fast rpb1 alleles that we identified did not result in significantly faster in vitro misincorporation rates than those resulting from rpb9Δ alone, and this epistasis is consistent with the idea that defects caused by the rpb1 alleles are related mechanistically to the defects caused by rpb9Δ. We conclude that Rpb9 supports intra-pol II interactions that modulate TL function and thus pol II enzymatic properties.
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http://dx.doi.org/10.1074/jbc.M116.714394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938204PMC
July 2016

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Mol Cell 2015 Jul;59(2):258-69

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA. Electronic address:

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.
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http://dx.doi.org/10.1016/j.molcel.2015.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643057PMC
July 2015

Uncoupling Promoter Opening from Start-Site Scanning.

Mol Cell 2015 Jul 11;59(1):133-8. Epub 2015 Jun 11.

Department of Structural Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.
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http://dx.doi.org/10.1016/j.molcel.2015.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490988PMC
July 2015

The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations.

Mol Cell 2015 Jun 7;58(6):1113-23. Epub 2015 May 7.

Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada; Département de Médecine, Faculté de Médecine, Université de Montréal, 2900 Boulevard Edouard-Montpetit, Montréal, QC H3T 1J4, Canada. Electronic address:

H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here we provide genomic and biochemical evidence that the RNA polymerase II (RNA Pol II) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nucleosome reassembly coupled to pervasive incorporation of H2A.Z by mislocalized SWR-C alters chromatin composition and contributes to cryptic initiation. Therefore, chaperone-mediated H2A.Z confinement is crucial for restricting the chromatin signature of gene promoters that otherwise may license or promote cryptic transcription.
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http://dx.doi.org/10.1016/j.molcel.2015.03.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475440PMC
June 2015

Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis.

Transcription 2014 ;5(3):e28869

a Department of Biochemistry and Biophysics; Texas A&M University; College Station, TX.

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn(2+) stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF.
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http://dx.doi.org/10.4161/trns.28869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574878PMC
October 2015

Relationships of RNA polymerase II genetic interactors to transcription start site usage defects and growth in Saccharomyces cerevisiae.

G3 (Bethesda) 2014 Nov 6;5(1):21-33. Epub 2014 Nov 6.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843

Transcription initiation by RNA Polymerase II (Pol II) is an essential step in gene expression and regulation in all organisms. Initiation requires a great number of factors, and defects in this process can be apparent in the form of altered transcription start site (TSS) selection in Saccharomyces cerevisiae (Baker's yeast). It has been shown previously that TSS selection in S. cerevisiae is altered in Pol II catalytic mutants defective in a conserved active site feature known as the trigger loop. Pol II trigger loop mutants show growth phenotypes in vivo that correlate with biochemical defects in vitro and exhibit wide-ranging genetic interactions. We assessed how Pol II mutant growth phenotypes and TSS selection in vivo are modified by Pol II genetic interactors to estimate the relationship between altered TSS selection in vivo and organismal fitness of Pol II mutants. We examined whether the magnitude of TSS selection defects could be correlated with Pol II mutant-transcription factor double mutant phenotypes. We observed broad genetic interactions among Pol II trigger loop mutants and General Transcription Factor (GTF) alleles, with reduced-activity Pol II mutants especially sensitive to defects in TFIIB. However, Pol II mutant growth defects could be uncoupled from TSS selection defects in some Pol II allele-GTF allele double mutants, whereas a number of other Pol II genetic interactors did not influence ADH1 start site selection alone or in combination with Pol II mutants. Initiation defects are likely only partially responsible for Pol II allele growth phenotypes, with some Pol II genetic interactors able to exacerbate Pol II mutant growth defects while leaving initiation at a model TSS selection promoter unaffected.
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http://dx.doi.org/10.1534/g3.114.015180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291466PMC
November 2014

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms.

Proc Natl Acad Sci U S A 2014 May 14;111(18):6642-7. Epub 2014 Apr 14.

Departments of Biology, Chemistry, Structural Biology, and Applied Physics, Stanford University, Stanford, CA 94305.

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.
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http://dx.doi.org/10.1073/pnas.1405181111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020062PMC
May 2014

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

RNA 2014 May 10;20(5):644-55. Epub 2014 Mar 10.

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.
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http://dx.doi.org/10.1261/rna.040444.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988566PMC
May 2014

Mechanisms of eukaryotic transcription.

Genome Biol 2013 ;14(9):311

A report on the Cold Spring Harbor Laboratory Mechanisms of Eukaryotic Transcription meeting, Cold Spring Harbor, New York, USA, August 27–31, 2013.
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http://dx.doi.org/10.1186/gb4132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053728PMC
January 2015

Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

Cell Rep 2013 Sep 29;4(5):974-84. Epub 2013 Aug 29.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0024, USA.

Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.
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http://dx.doi.org/10.1016/j.celrep.2013.07.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801175PMC
September 2013

From structure to systems: high-resolution, quantitative genetic analysis of RNA polymerase II.

Cell 2013 Aug 8;154(4):775-88. Epub 2013 Aug 8.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ∼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.
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http://dx.doi.org/10.1016/j.cell.2013.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932829PMC
August 2013

Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae.

Authors:
Craig D Kaplan

Biochim Biophys Acta 2013 Jan 26;1829(1):39-54. Epub 2012 Sep 26.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.

Transcription by RNA polymerase II (Pol II), and all RNA polymerases for that matter, may be understood as comprising two cycles. The first cycle relates to the basic mechanism of the transcription process wherein Pol II must select the appropriate nucleoside triphosphate (NTP) substrate complementary to the DNA template, catalyze phosphodiester bond formation, and translocate to the next position on the DNA template. Performing this cycle in an iterative fashion allows the synthesis of RNA chains that can be over one million nucleotides in length in some larger eukaryotes. Overlaid upon this enzymatic cycle, transcription may be divided into another cycle of three phases: initiation, elongation, and termination. Each of these phases has a large number of associated transcription factors that function to promote or regulate the gene expression process. Complicating matters, each phase of the latter transcription cycle are coincident with cotranscriptional RNA processing events. Additionally, transcription takes place within a highly dynamic and regulated chromatin environment. This chromatin environment is radically impacted by active transcription and associated chromatin modifications and remodeling, while also functioning as a major platform for Pol II regulation. This review will focus on our basic knowledge of the Pol II transcription mechanism, and how altered Pol II activity impacts gene expression in vivo in the model eukaryote Saccharomyces cerevisiae. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.
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http://dx.doi.org/10.1016/j.bbagrm.2012.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026157PMC
January 2013

Dissection of Pol II trigger loop function and Pol II activity-dependent control of start site selection in vivo.

PLoS Genet 2012 12;8(4):e1002627. Epub 2012 Apr 12.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.
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http://dx.doi.org/10.1371/journal.pgen.1002627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325174PMC
September 2012

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II.

Proc Natl Acad Sci U S A 2012 Apr 9;109(17):6555-60. Epub 2012 Apr 9.

Biophysics Program, Stanford University, Stanford, CA 94305, USA.

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.
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http://dx.doi.org/10.1073/pnas.1200939109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340090PMC
April 2012

Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.

Proc Natl Acad Sci U S A 2012 Mar 12;109(13):4816-21. Epub 2012 Mar 12.

Department of Structural Biology, Stanford University, Stanford, CA 94305, USA.

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.
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http://dx.doi.org/10.1073/pnas.1201448109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323989PMC
March 2012

Competing for the clamp: promoting RNA polymerase processivity and managing the transition from initiation to elongation.

Mol Cell 2011 Jul;43(2):161-3

Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA.

Transcription elongation factor NusG/Spt5 spans the central cleft of RNA polymerase and functionally competes with transcription initiation factors. This work highlights the RNA polymerase clamp as a target for regulation and points to dynamic interactions between initiation and elongation machineries.
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http://dx.doi.org/10.1016/j.molcel.2011.07.002DOI Listing
July 2011