scholarly journals The bacterial enhancer-dependent RNA polymerase

Biochemical Journal â—½  
2016 â—½  
Vol 473 (21) â—½  
pp. 3741-3753 â—½  
Author(s):  
Nan Zhang â—½  
Vidya C. Darbari â—½  
Robert Glyde â—½  
Xiaodong Zhang â—½  
Martin Buck
Keyword(s):  
Gene Expression â—½  
Rna Polymerase â—½  
Self Assembly â—½  
Transcription Initiation â—½  
Large Scale â—½  
Bacterial Cells â—½  
Transcription Control â—½  
Amino Terminal â—½  
Promoter Dna â—½  
Regulated Gene Expression

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

scholarly journals Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

eLife â—½  
10.7554/elife.50928 â—½  
2019 â—½  
Vol 8 â—½  
Author(s):  
Juncao Xu â—½  
Kaijie Cui â—½  
Liqiang Shen â—½  
Jing Shi â—½  
Lingting Li â—½  
...  
Keyword(s):  
Rna Polymerase â—½  
Transcription Initiation â—½  
Transcription Activation â—½  
Initiation Factor â—½  
Bacterial Cells â—½  
E Coli â—½  
Exchange Mass Spectrometry â—½  
Transcription Initiation Factor â—½  
Hydrogen Deuterium â—½  
Promoter Dna

σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase–promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

Nutrient-regulated gene expression in eukaryotes

10.1042/bss0730085 â—½  
2006 â—½  
Vol 73 â—½  
pp. 85-96 â—½  
Author(s):  
Richard J. Reece â—½  
Laila Beynon â—½  
Stacey Holden â—½  
Amanda D. Hughes â—½  
Karine Rébora â—½  
...  
Keyword(s):  
Gene Expression â—½  
Rna Polymerase â—½  
Rna Polymerase Ii â—½  
Transcriptional Control â—½  
Direct Interaction â—½  
Signalling Pathways â—½  
A Cell â—½  
One Step â—½  
Higher Eukaryotes â—½  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 â—½  
Vol 50 (4) â—½  
pp. 909-920 â—½  
Author(s):  
Iwona K Kolasa â—½  
Tomasz Łoziński â—½  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli â—½  
Rna Polymerase â—½  
Transcription Initiation â—½  
Specific Interaction â—½  
Structural Data â—½  
Structural Morphology â—½  
Promoter Dna â—½  
Sigma Subunit â—½  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

scholarly journals Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

2020 â—½  
Author(s):  
Brent De Wijngaert â—½  
Shemaila Sultana â—½  
Chhaya Dharia â—½  
Hans Vanbuel â—½  
Jiayu Shen â—½  
...  
Keyword(s):  
Rna Polymerase â—½  
Transcription Initiation â—½  
Large Scale â—½  
Rna Synthesis â—½  
Mitochondrial Rna â—½  
Initiation Complex â—½  
Mitochondrial Rna Polymerase â—½  
Catalytically Active â—½  
Promoter Melting â—½  
Antiviral Nucleosides

Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the −4 to −2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPαS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 â—½  
Vol 11 (1) â—½  
Author(s):  
Sung-Hoon Jun â—½  
Jaekyung Hyun â—½  
Jeong Seok Cha â—½  
Hoyoung Kim â—½  
Michael S. Bartlett â—½  
...  
Keyword(s):  
Dna Binding â—½  
Rna Polymerase â—½  
Transcription Initiation â—½  
Structural Basis â—½  
Transcription Bubble â—½  
Cryo Electron Microscopy â—½  
Direct Binding â—½  
Binding Cleft â—½  
Promoter Dna â—½  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

scholarly journals A Large Scale Genetic Analysis of c-Myc-regulated Gene Expression Patterns

2003 â—½  
Vol 278 (14) â—½  
pp. 12563-12573 â—½  
Author(s):  
Brenda C. O'Connell â—½  
Ann F. Cheung â—½  
Carl P. Simkevich â—½  
Wanny Tam â—½  
Xiaojia Ren â—½  
...  
Keyword(s):  
Gene Expression â—½  
Genetic Analysis â—½  
Large Scale â—½  
Expression Patterns â—½  
Gene Expression Patterns â—½  
Regulated Gene Expression

scholarly journals An Archaeal Fluoride-Responsive Riboswitch Provides an Inducible Expression System for Hyperthermophiles

2018 â—½  
Vol 84 (7) â—½  
Author(s):  
Michael Clayton Speed â—½  
Brett W. Burkhart â—½  
Jonathan W. Picking â—½  
Thomas J. Santangelo
Keyword(s):  
Gene Expression â—½  
Transcription Initiation â—½  
Expression System â—½  
Regulatory Control â—½  
Cell Physiology â—½  
Content Type â—½  
Thermococcus Kodakarensis â—½  
Export Pathway â—½  
Regulated Expression â—½  
Regulated Gene Expression

ABSTRACT Robust genetic systems for the hyperthermophilic Thermococcales have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a cis -encoded functional riboswitch in the model archaeon Thermococcus kodakarensis . Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates T. kodakarensis strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles. IMPORTANCE Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in Archaea is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in Bacteria and Eukarya , but seemingly absent in Archaea . We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon Thermococcus kodakarensis . The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in Thermococcus kodakarensis , demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 â—½  
Vol 117 (7) â—½  
pp. 3560-3567 â—½  
Author(s):  
Daniel J. Luciano â—½  
Joel G. Belasco
Keyword(s):  
Rna Polymerase â—½  
Transcription Initiation â—½  
Promoter Sequence â—½  
Rna Degradation â—½  
Initiation Site â—½  
Bacterial Cells â—½  
E Coli â—½  
N Incorporation â—½  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 â—½  
Vol 24 (7) â—½  
pp. 2863-2874 â—½  
Author(s):  
Thomas C. Tubon â—½  
William P. Tansey â—½  
Winship Herr
Keyword(s):  
Rna Polymerase â—½  
Rna Polymerase Ii â—½  
Transcription Initiation â—½  
Terminal Region â—½  
General Role â—½  
Pol Ii â—½  
Amino Terminal â—½  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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