Biology subjects

Nickels, B. E.

Publications and source records attributed to Nickels, B. E..

13 recordsLinked to original sources

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↗

bioRxiv2022

Structural basis of transcription antitermination by Q{lambda}: NusA induces refolding of Q{lambda} to form nozzle for RNA polymerase exit channel

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein from bacteriophage 21 (Q21) functions by forming a "nozzle" that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein from bacteriophage {lambda} (Q{lambda}), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Q{lambda}, the Q{lambda}-QBE complex, the NusA-free "pre-engaged" Q{lambda}-loading complex, and the NusA-containing "engaged" Q{lambda}-loading complex. The results show that Q{lambda}, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Q{lambda} has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Q{lambda} to form a "pre-engaged" loading complex, followed by NusA-facilitated refolding of Q{lambda} to form an "engaged" loading complex. The results establish Q{lambda} and Q21 are not structural homologs and are solely functional analogs. SIGNIFICANCE STATEMENTBacteriophage Q proteins are textbook examples of regulators of gene expression that function at the level of transcription antitermination. Here, we report structures defining the mechanism of antitermination by the Q protein of bacteriophage {lambda} (Q{lambda}). The results show Q{lambda} forms a "nozzle" that narrows and extends the RNA polymerase RNA-exit channel, precluding the formation of terminator RNA hairpins. The results show Q{lambda} exhibits no structural similarity to the Q protein of bacteriophage 21 (Q21), employs a different mechanism for DNA binding than Q21, and employs a more complex process of loading onto RNA polymerase than Q21. We conclude Q{lambda} and Q21 are not structural homologs and are solely functional analogs, akin to a bird wing and a bat wing.

molecular biology↗

bioRxiv2022

Structural and mechanistic basis of σ-dependent transcriptional pausing

In {sigma}-dependent transcriptional pausing, the transcription initiation factor {sigma}, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in {sigma}-dependent pausing, the RNAP active center can access off-pathway "backtracked" states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway "scrunched" states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of {sigma} relative to DNA at the{lambda} PR promoter, we show directly that {sigma}-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that {sigma}-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 ([~]16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3 end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in {sigma}-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching.

biochemistry↗

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

Structural and mechanistic basis of RNA extension in reiterative transcription initiation: RNA slippingwithout DNA scrunching

Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5 sequences non-complementary to the DNA template. Here, using crystallography and cryo-EM to define structures, protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation: (1) occurs without DNA scrunching, (2) involves a short, 2-3 bp RNA-DNA hybrid, and (3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit.

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

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

Chemical modifications of RNA 5' ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates (NTPs) for use as initiating entities, providing an ab initio mechanism for altering the RNA 5' end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here we use massively systematic transcript end readout ("MASTER") to detect and quantify RNA 5' ends generated by primer-dependent initiation for ~410 (~1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position, but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

biochemistry↗

bioRxiv2020

A Spatiotemporal Molecular Switch Governs Plant Asymmetric Cell Division

Asymmetric cell division (ACD) often requires protein polarization in the mother cell to produce daughter cells with distinct identities ("cell-fate asymmetry"). Here, we define a previously undocumented mechanism for establishing cell-fate asymmetry in Arabidopsis stomatal stem cells. In particular, we show that polarization of BSL protein phosphatases promotes stomatal ACD by establishing a "kinase-based signaling asymmetry" in the two daughter cells. BSL polarization in the stomatal ACD mother cell is triggered upon commitment to cell division. Polarized BSL is inherited by the differentiating daughter cell where it suppresses cell division and promotes cell-fate determination. Plants lacking BSL exhibit stomatal over-proliferation, demonstrating BSL plays an essential role in stomatal development. Our findings establish that BSL polarization provides a spatiotemporal molecular switch that enables cell-fate asymmetry in stomatal ACD daughter cells. We propose BSL polarization is triggered by an ACD "checkpoint" in the mother cell that monitors establishment of division-plane asymmetry.

plant biology↗

bioRxiv2019

XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines RNAP-active-center position relative to DNA in vivo with single-nucleotide resolution (XACT-seq; crosslink-between-active-center-and-template sequencing). We apply this method to detect and quantify pausing in initial transcription at 411 ([~]4,000,000) promoter sequences in vivo, in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4-5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard-coded by sequence elements similar to those for transcription-elongation pausing.

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↗

bioRxiv2019

Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves \"adaptation,\" the integration of ~30-bp DNA fragments into the CRISPR array as \"spacer\" sequences, and \"interference,\" the targeted degradation of DNA containing a \"protospacer\" sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1-4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with \"primed adaptation,\" ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas \"self-targeting\" locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, \"FragSeq,\" and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

microbiology↗

bioRxiv2018

Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

biochemistry↗

bioRxiv2017

“CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

Nucleoside-containing metabolites such as NAD+ can be incorporated as \"5' caps\" on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report \"CapZyme-Seq,\" a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

biochemistry↗