Jiao, X, Doamekpor S, Bird JG, Nickels BE, Tong L, Hart RP, Kiledjian M.  2017.  5′-end Nicotinamide Adenine Dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding.. Cell. 168(6):1015-1027.
Garner, AL, Rammohan J, Huynh JP, Onder LM, Chen J, Bae B, Jensen D, Weiss LA, Manzano AR, Darst SA et al..  2017.  Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence. Journal of Bacteriology. 199:e00698-16..
Vvedenskaya, IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE.  2016.  Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.. Proc. Natl. Acad. Sci. U.S.A. 113(21):E2899-2905.
Bird, JG, Zhang Y, Tian Y, Panova N, Barvík I, Greene L, Liu M, Buckley B, Krásný L, Lee JK et al..  2016.  The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA. Nature. 535(7612):444-447.
Winkelman, JT, Vvedenskaya IO, Zhang Y, Zhang Y, Bird JG, Taylor DM, Gourse RL, Ebright RH, Nickels BE.  2016.  Multiplexed protein-DNA crosslinking: scrunching in transcription start site selection. Science. 351(6277):1090-1093.
Schifano, JM, Cruz JW, Edifor R, Vvedenskaya IO, Ouyang M, Husson RN, Nickels BE, Woychik NA.  2016.  tRNA is a new target for cleavage by a MazF toxin. Nucleic Acids Research. 44(3):1256-1270.
Druzhinin, SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE.  2015.  A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq. PLoS Genet. 11(7):e1005348.
Cruz, JW, Sharp JD, Hoffer ED, Maehigashi T, Vvedenskaya IO, Konkimalla A, Husson RN, Nickels BE, Dunham C, Woychik NA.  2015.  Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun. 6:7480.
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively systematic transcript end readout, “MASTER”: transcription start site selection, transcriptional slippage, and transcript yields. Molecular Cell. 60:953-965.
Vvedenskaya, IO, Goldman SR, Nickels BE.  2015.  Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5' ends. Methods Mol Biol. 1276:211-228.
Goldman, SR, Nair N, Wells C, Nickels BE, Hochschild A.  2015.  The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo. eLife. 4:e10514.
Ramsey, KM, Osborne ML, Vvedenskaya IO, Su C, Nickels BE, Dove SL.  2015.  Ubiquitous promoter-localization of essential virulence regulators in Francisella tularensis.. PLoS Pathog. 11(4):e1004793.
Vvedenskaya, IO, Vahedian-Movahed H, Bird JG, Knoblauch JG, Goldman SR, Zhang Y, Ebright RH, Nickels BE.  2014.  Interactions between RNA polymerase and the "core recognition element" counteract pausing. Science. 344(6189):1285-1289. Abstract
Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.
Schifano, JM, Vvedenskaya IO, Knoblauch JG, Ouyang M, Nickels BE, Woychik NA.  2014.  An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3.. Nature Communications. 5:1–11. Abstract
Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.
Vorobiev, SM, Gensler Y, Vahedian-Movahed H, Seetharaman J, Su M, Huang JY, Xiao R, Kornhaber G, Montelione GT, Tong L et al..  2014.  Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor λQ. Structure. 22:488-495.
Bao, X, Nickels BE, Fan H.  2012.  Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of sigma66. Proc Natl Acad Sci U S A. AbstractWebsite
The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. Primary, or housekeeping, sigma factors are responsible for most of the gene expression that occurs during the exponential phase of growth. Primary sigma factors share four regions of conserved sequence, regions 1-4, which have been further subdivided. Many primary sigma factors also contain a nonconserved region (NCR) located between subregions 1.2 and 2.1, which can vary widely in length. Interactions between the NCR of the primary sigma factor of Escherichia coli, sigma(70), and the beta' subunit of the E. coli core enzyme have been shown to influence gene expression, suggesting that the NCR of primary sigma factors represents a potential target for transcription regulation. Here, we report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). We demonstrate in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of sigma(66)-dependent promoters. We further show that GrgA activates transcription by contacting the NCR of the primary sigma factor of C. trachomatis, sigma(66). Our findings suggest GrgA serves as an important regulator of sigma(66)-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, our findings highlight how nonconserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.
Weiss, LA, Harrison PG, Nickels BE, Glickman MS, Campbell EA, Darst SA, Stallings CL.  2012.  Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis. J Bacteriol. 194:5621-5631. AbstractWebsite
Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.
Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L.  2012.  A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.. Nucleic Acids Research. Abstract
Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstract
Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Nickels, BE.  2012.  A new way to start: nanoRNA-mediated priming of transcription initiation.. Transcription. 3(6):300-304. Abstract
A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.
Bao, X, Pachikara N, Oey CB, Balakrishnan A, Westblade LF, Tan M, Chase T, Nickels BE, Fan H.  2011.  Noncoding Nucleotides and Amino Acids near the Active Site Regulate Peptide Deformylase Expression and Inhibitor Susceptibility in Chlamydia trachomatis. Microbiology. AbstractWebsite
Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the o66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability.
Goldman, SR, Sharp JS, Vvedenskaya IO, Livny J, Dove SL, Nickels BE.  2011.  NanoRNAs Prime Transcription Initiation In Vivo. Mol Cell. 42:817-825. AbstractWebsite
It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to approximately 4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.
Nickels, BE, Dove SL.  2011.  NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria. J Mol Biol. AbstractWebsite
It has been widely assumed that all transcription in cells occur using NTPs only (i.e., de novo). However, it has been known for several decades that both prokaryotic and eukaryotic RNA polymerases can utilize small (2 to approximately 5 nt) RNAs to prime transcription initiation in vitro, raising the possibility that small RNAs might also prime transcription initiation in vivo. A new study by Goldman et al. has now provided the first evidence that priming with so-called "nanoRNAs" (i.e., 2 to approximately 5 nt RNAs) can, in fact, occur in vivo. Furthermore, this study provides evidence that altering the extent of nanoRNA-mediated priming of transcription initiation can profoundly influence global gene expression. In this perspective, we summarize the findings of Goldman et al. and discuss the prospect that nanoRNA-mediated priming of transcription initiation represents an underappreciated aspect of gene expression in vivo.
Twist, KA, Husnain SI, Franke JD, Jain D, Campbell EA, Nickels BE, Thomas MS, Darst SA, Westblade LF.  2011.  A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the alpha C-terminal domain. Protein Sci. 20:986-95. AbstractWebsite
The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric beta or beta' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric alpha-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the alpha C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any alpha-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.
Deighan, P, Pukhrambam C, Nickels BE, Hochschild A.  2011.  Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex. Genes Dev. 25:77-88. AbstractWebsite
The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) in complex with a sigma factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between sigma and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of sigma upon the transition to elongation. Furthermore, sigma can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with sigma also function to increase the sigma content of downstream elongation complexes. Our findings establish sigma-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.