Biology subjects

Kaplan, C. D.

Publications and source records attributed to Kaplan, C. D..

16 recordsLinked to original sources

bioRxiv2026

Single-molecule nanopore-tweezers analysis of transcription elongation by intact and "stalk-less" yeast RNA polymerase II

Single-molecule picometer-resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic-acid motor protein on a nucleic-acid track with sequence registration, sub-nucleotide spatial resolution, sub-millisecond temporal resolution, and the ability to apply forces that assist or oppose translocation. Recently, we used SPRNT to analyze the translocation of single molecules of Escherichia coli RNA polymerase relative to the DNA template strand during transcription elongation, and we directly detected sequence-dependent pausing and formation of a "half-translocated state" at the E. coli yrbL consensus pause element. Here, we apply SPRNT to analyze the translocation of single molecules of yeast RNA polymerase II (Pol II) relative to the DNA template strand during transcription elongation with single-nucleotide spatial resolution and millisecond-scale temporal resolution at biologically relevant, saturating substrate concentrations; we compare translocation by intact, 12-subunit Pol II to translocation by a 10-subunit Pol II sub-assembly lacking the dissociable Rpb4-Rpb7 Pol II "stalk"; and we assess possible pausing by intact Pol II and stalk-less Pol II at the E. coli yrbL consensus pause element. The results show that intact Pol II elongates more rapidly than stalk-less Pol II and show that neither intact Pol II nor stalk-less Pol II pauses at the E. coli yrbL consensus pause element. One-sentence summaryNanopore tweezers enable monitoring of translocation of RNA polymerase II relative to DNA in transcription elongation with single-nucleotide spatial resolution and millisecond-scale temporal resolution at biologically relevant, saturating substrate concentrations.

molecular biology↗

bioRxiv2025

RNA polymerase II-TFIIE-TFIIH interface functions in transcription start site selection in Saccharomyces cerevisiae

During transcription initiation in Saccharomyces cerevisiae, RNA polymerase II (Pol II) and general transcription factors (GTFs) assemble upstream of transcription start sites (TSSs) to form the pre-initiation complex (PIC). In this model organism, yeast, the PIC selects TSSs through a unidirectional scanning mechanism referred to as promoter scanning. Previous studies have shown that the TFIIH subunit Tfb3 connects TFIIH to the rest of the PIC through interactions with Pol II and the GTF TFIIE. Activities within the PIC that influence TSS selection can do so by control of initiation efficiency at individual TSSs or by control of TSS scanning (either rate of scanning or scanning processivity). To understand how this critical interface withing the PIC participates in scanning, we used genetic screens to identify tfb3 and tfa1 mutants that alter initiation using initiation-linked phenotypes. We found mutations within the TFIIH-Pol II-TFIIE interface able to alter promoter scanning in either upstream or downstream directions, suggesting that changes to this interface can fine-tune scanning. Subsets of alleles were analyzed using TSS sequencing approaches, showing that tested tfb3 and tfa1 alleles shift TSS distributions across most genomic promoters. Genetic interaction and genomic analysis revealed that the Tfb3 interfaces with Rpb7 and Tfa1 separately contribute to promoter scanning, and that tfb3 alleles exhibit additive effects with scanning processivity mutants in, consistent with Tfb3-PIC interactions modulating scanning processivity. The ability of this interface to easily modulate scanning in both directions is consistent with the types of changes that might incrementally allow promoter scanning to have evolved.

molecular biology↗

bioRxiv2025

Genetic dissection of transcription start site selection by RNA polymerase II in Saccharomyces cerevisiae

Transcription initiation by RNA Pol II is driven by a preinitiation complex (PIC) comprising Pol II and general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. In Saccharomyces cerevisiae, transcription start site (TSS) selection proceeds by a promoter scanning mechanism where the PIC scans downstream for TSSs. To determine which factors participate in TSS selection by promoter scanning, we designed and implemented forward genetic selections using initiation-sensitive genetic reporters. These reporters were of two types, either being sensitive to upstream or downstream TSS shifts, allowing detection of alleles that affect promoter scanning in different directions. From >1000 candidates, we identified three primary classes of mutants: existing and novel mutants within known PIC components, including novel alleles of multiple TFIIH subunits; mutants that alter promoter scanning indirectly through cellular conditions (GTP levels and Mn2+ levels); and mutants in chromatin and transcription elongation-related factors. Genome-wide analysis using TSS mapping shows that tested PIC mutants alter TSS usage globally across promoters while tested chromatin and transcription elongation mutants had much more limited effects. These studies expand the spectrum of mutants able to perturb initiation by promoter scanning and suggest potential plasticity in the TFIIH structure that may be part of the evolution of this initiation mechanism.

molecular biology↗

bioRxiv2025

Substitutions in RNA-binding protein Hrp1 map a potential interaction surface with the yeast RNA polymerase II elongation complex

Anti-termination factors for eukaryotic RNA polymerase II (RNAP II) that are released upon binding sequences in the terminator of nascent transcripts were proposed almost 40 years ago but few candidates have been found. Here we report genetic evidence that the yeast nuclear RNA-binding protein Hrp1, also known as Nab4 and CF1B, acts as an RNAP II anti-termination factor. A Lys to Glu substitution at residue 9 (K9E) of the Rpb3 subunit of RNAP II causes readthrough of Nrd1-Nab3-Sen1-dependent (NNS) terminators and cold-sensitive growth, as does Asp but not Ala, Met, Arg, or Gln substitution. These allele-specific phenotypes and the location of Rpb3-K9 suggests substitution with Glu or Asp stabilizes binding of an anti-termination factor via a salt bridge. A genome-wide selection for suppressors of the cold-sensitivity of Rpb3-K9E yielded an Arg to Gly substitution at residue 317 of Hrp1 in RNA recognition motif 2 (RRM2), consistent with the hypothesis. A targeted selection for suppressors of Rpb3-K9E in HRP1 yielded substitutions in RRMs 1 and 2, an essential Met- and Gln-rich region C-terminal to the RRMs, and other mutations. We propose that Hrp1 binds to the RNAP II elongation complex via these regions to promote elongation and, in the presence of Rpb3-K9E, is less rapidly released upon binding terminator sequences in the nascent transcript, resulting in terminator readthrough. The Rpb3-K9E-suppressor substitutions in Hrp1 likely weaken binding to the RNAP II elongation complex, compensating for Rpb3-K9E.

molecular biology↗

bioRxiv2024

Higher-order epistasis within Pol II trigger loop haplotypes

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL for the purpose of understand functional interactions between residues and to understand how individual mutants might alter TL function. We identified widespread incompatibility between TLs of different species when placed in the Saccharomyces cerevisiae Pol II context, indicating species-specific interactions between otherwise highly conserved TLs and its surroundings. These interactions represent epistasis between TL residues and the rest of Pol II. We sought to understand why certain TL sequences are incompatible with S. cerevisiae Pol II and to dissect the nature of genetic interactions within multiply substituted TLs as a window on higher order epistasis in this system. We identified both positive and negative higher-order residue interactions within example TL haplotypes. Intricate higher-order epistasis formed by TL residues was sometimes only apparent from analysis of intermediate genotypes, emphasizing complexity of epistatic interactions. Furthermore, we distinguished TL substitutions with distinct classes of epistatic patterns, suggesting specific TL residues that potentially influence TL evolution. Our examples of complex residue interactions suggest possible pathways for epistasis to facilitate Pol II evolution.

genetics↗

bioRxiv2023

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 A using a free-electron laser

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

biophysics↗

bioRxiv2023

Widespread epistasis shapes RNA Polymerase II active site function and evolution

Multi-subunit RNA Polymerases (msRNAPs) are responsible for transcription in all kingdoms of life. These enzymes rely on dynamic, highly conserved active site domains such as the so-called "trigger loop" (TL) to accomplish steps in the transcription cycle. Mutations in the RNA polymerase II (Pol II) TL confer a spectrum of biochemical and genetic phenotypes that suggest two main classes, which decrease or increase catalysis or other nucleotide addition cycle (NAC) events. The Pol II active site relies on networks of residue interactions to function, and mutations likely perturb these networks in ways that may alter mechanisms. We have undertaken a structural genetics approach to reveal residue interactions within and surrounding the Pol II TL - determining its "interaction landscape" - by deep mutational scanning in Saccharomyces cerevisiae Pol II. This analysis reveals connections between TL residues and surrounding domains, demonstrating that TL function is tightly coupled to its specific enzyme context.

genetics↗

bioRxiv2022

Characterization of RNA Polymerase II Trigger Loop Mutations using Molecular Dynamics Simulations and Machine Learning

Catalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that lethal mutations tended to increase distances of TL residues to the NTP substrate, while viable loss-of-function (LOF) substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, GOF mutants generally have a disrupting effect on hydrophobic contacts among TL and nearby helices. AUTHOR SUMMARYRNA polymerase II (Pol II) synthesizes RNA with the help of an active site domain called trigger loop (TL). The mutations of TL cause changes in the activity of Pol II that could range from gain-of function (GOF) to loss-of-function (LOF) or lethal. This study provides a systematic characterization of the structural and functional outcomes of the TL mutations using molecular dynamics (MD) simulations and machine learning (ML). We obtained functional phenotypes of mutants by ML using the genetic fitness scores as the input. We revealed that mutant TL sequences could predict the functional outcomes at a relatively high correlation. Then, we performed MD simulations to relate the structural information to the phenotypes. The analysis of the MD data suggested that the lethal mutants had increased distances between the TL and the substrate, while a subset of LOF mutants showed increased distances between TL and another active site domain called bridge helix (BH). On the other hand, GOF mutants had effects on the hydrophobic interactions around the active site. Overall, this study enhances our understanding of the effects of TL mutations to the Pol II function.

biophysics↗

bioRxiv2022

No evidence that synonymous mutations in yeast genes are mostly deleterious

In a recent paper1, Shen et al. reported that most mutations in the coding regions of 21 yeast genes were strongly deleterious, and that the distributions of fitness effects were similar for synonymous and nonsynonymous mutations. Taken at face value, these results would conflict with well-established findings from a broad range of fields and approaches. Here, we argue that these results arose from a lack of appropriate controls for the impacts of background genetic effects in edited strains. A re-examination of the data in Shen et al. strongly suggests that it is entirely consistent with the expectation that most nonsynonymous and nearly all synonymous mutations have no detectable effects on fitness. We present analyses which show that the data is inconsistent with the proposed explanation that pervasive fitness effects of synonymous mutations arise from their effects on mRNA levels, that the sequence-based fitness assay overestimates fitness effects compared to the growth-based fitness assay, and that the observed wide fitness distributions for nonsense mutations are consistent with off-target effects or other uncontrolled sources of biological variation contributing to measured fitness. We conclude by discussing the essential controls and other experimental design considerations that are required to produce interpretable results regarding the fitness effects of mutations in large-scale screens.

genetics↗

bioRxiv2021

Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae determines contributions of DNA sequence and RNA Polymerase II activity

Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here, we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, NTP substrate levels, and RNA polymerase II (Pol II) activity to TSS selection by "promoter scanning" in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1, and +1 positions quantitatively in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to GTP levels at the first five transcript positions and to CTP and UTP levels at the second position genome wide. These results suggest individual NTP levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.

molecular biology↗

bioRxiv2021

Thiolutin is a direct inhibitor of RNA Polymerase II

Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutins mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.

molecular biology↗

bioRxiv2021

Ssl2/TFIIH Function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window [~]40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

molecular biology↗

bioRxiv2021

Structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in Nucleotide Excision Repair

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit core TFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 [A] resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3 and 5 side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 would extend the DNA opening at the lesion and deliver the damaged strand to Rad3 (XPD) in an unwound form suitable for subsequent lesion scanning and verification.

biophysics↗

bioRxiv2021

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

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 characterization of genes that regulate mitochondrial copper homeostasis is lacking. To identify genes required for mitochondrial copper homeostasis, we performed a genome-wide copper-sensitized screen using DNA barcoded yeast deletion library. Our screen recovered a number of genes known to be involved in cellular copper homeostasis while revealing 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 the Rim mutants impact mitochondrial CcO function by maintaining vacuolar acidity. 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 a number of novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.

biochemistry↗

bioRxiv2019

Promoter scanning during transcription initiation in Saccharomyces cerevisiae: Pol II in the \"shooting gallery\"

BackgroundThe 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 S. cerevisiae, a preinitiation complex comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the preinitiation complex (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. ResultsTo determine extent of promoter scanning across promoter classes in S. cerevisiae, we perturbed Pol II catalytic activity and GTF function and analyzed 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 extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. ConclusionsOur 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.

molecular biology↗

bioRxiv2018

Perturbations of transcription and gene expression-associated processes alter distribution of cell size values in Saccharomyces cerevisiae

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 Saccharomyces cerevisiae 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 Saccharomyces cerevisiae. 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.

genetics↗