scholarly journals Structural basis for transcription initiation by bacterial ECF σ factors

2019 ◽  
Author(s):  
Lingting Li ◽  
Chengli Fang ◽  
Ningning Zhuang ◽  
Tiantian Wang ◽  
Yu Zhang
Keyword(s):  
Crystal Structure ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Specific Gene ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Transcription Initiation Complex ◽  
Promoter Dna ◽  
Context Specific

AbstractBacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

scholarly journals Bacterial RNA polymerase can retain σ70 throughout transcription

2016 ◽  
Vol 113 (3) ◽  
pp. 602-607 ◽  
Author(s):  
Timothy T. Harden ◽  
Christopher D. Wells ◽  
Larry J. Friedman ◽  
Robert Landick ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Messenger Rna ◽  
Initiation Factors ◽  
Bacterial Rna ◽  
Direct Measurements ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Transcript Elongation

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–RNAP complex during initiation from the λ PR′ promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ70 during transcription.

scholarly journals X-ray crystal structure of a reiterative transcription complex reveals an atypical RNA extension pathway

2017 ◽  
Vol 114 (31) ◽  
pp. 8211-8216 ◽  
Author(s):  
Katsuhiko S. Murakami ◽  
Yeonoh Shin ◽  
Charles L. Turnbough ◽  
Vadim Molodtsov
Keyword(s):  
Crystal Structure ◽  
Transcription Initiation ◽  
Thermus Thermophilus ◽  
Elongation Complex ◽  
Nascent Transcript ◽  
Bacterial Enzyme ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Reiterative Transcription ◽  
Nascent Transcripts

Reiterative transcription is a noncanonical form of RNA synthesis in which a nucleotide specified by a single base in the DNA template is repetitively added to the nascent transcript. Here we determined the crystal structure of an RNA polymerase, the bacterial enzyme from Thermus thermophilus, engaged in reiterative transcription during transcription initiation at a promoter resembling the pyrG promoter of Bacillus subtilis. The structure reveals that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme, thereby allowing RNA extension without displacement of the promoter recognition σ-factor. Nascent transcripts containing reiteratively added G residues are eventually extended by nonreiterative transcription, revealing an atypical pathway for the formation of a transcription elongation complex.

scholarly journals Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase

2019 ◽  
Vol 116 (38) ◽  
pp. 18923-18927 ◽  
Author(s):  
Alexis Jaramillo Cartagena ◽  
Amy B. Banta ◽  
Nikhil Sathyan ◽  
Wilma Ross ◽  
Richard L. Gourse ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Functional Importance ◽  
Structural Element ◽  
Cryo Electron Microscopy ◽  
Promoter Complex ◽  
Σ Factor ◽  
Promoter Dna ◽  
Transcription Activators

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti–σ-factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS-regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that we call the β′-clamp-toe (β′CT). Deletion of the β′CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β′CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS2 and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

scholarly journals Structures and mechanism of transcription initiation by bacterial ECF factors

2019 ◽  
Vol 47 (13) ◽  
pp. 7094-7104 ◽  
Author(s):  
Chengli Fang ◽  
Lingting Li ◽  
Liqiang Shen ◽  
Jing Shi ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Active Center ◽  
Transcription Initiation ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Specific Gene ◽  
Initiation Complex ◽  
E Coli ◽  
Promoter Escape ◽  
Promoter Recognition ◽  
Transcription Initiation Complex

Abstract Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE—the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

scholarly journals Structural Basis of ECF-σ-Factor-Dependent Transcription Initiation

2018 ◽  
Author(s):  
Wei Lin ◽  
Sukhendu Mandal ◽  
David Degen ◽  
Min Sung Cho ◽  
Yu Feng ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Crystal Structures ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Functional Modules ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Σ Factor ◽  
Promoter Dna

SUMMARYExtracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of the six conserved functional modules present in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor--although unrelated in sequence--follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent transcription initiation.

scholarly journals Structural studies of Mot1 and NC2 in transcription initiation process

2014 ◽  
Vol 70 (a1) ◽  
pp. C1396-C1396
Author(s):  
Agata Butryn ◽  
Jan Schuller ◽  
Gabriele Stöhr ◽  
Friedrich Förster ◽  
Karl-Peter Hopfner
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Promoter Regions ◽  
Protein Nucleic Acid ◽  
Promoter Dna ◽  
Tata Box Binding Protein

Regulation of protein–nucleic acid interactions plays a key role in various cellular processes. The SNF2/SWI2 protein family forms a large and diverse class of proteins and multiprotein assemblies, which use energy derived from ATP hydrolysis to disrupt or modify protein-DNA interactions. They possess a conserved helicase-like domain accompanied by protein-specific targeting and regulation regions. The SNF2/SWI2 family member Mot1 (Modifier of transcription 1, also known as BTAF1) is a single polypeptide ATP-dependent remodeler. Mot1 acts directly on TBP (TATA-box binding protein) regulating RNA polymerase II preinitiation complex formation in the first stages. Global distribution of TBP on promoter regions is also modulated by NC2 (Negative Cofactor 2). It has been suggested that Mot1 and NC2 can co occupy the same promoter sites influencing the assembly of the transcription machinery simultaneously. Our understanding of the molecular mechanism of SWI2/SNF2 family ATPases is very limited. Previously, we have reported the crystal structure of the complex of Encephalitozoon cuniculi N terminal domain of Mot1 (Mot1NTD) with its substrate, TBP [1]. Here we present the crystal structure of the TBP NC2 Mot1NTD complex bound to a promoter DNA fragment at 3.9Å resolution. In our studies we have applied a combined structural biology approach using crystallography, electron microscopy reconstructions and chemical cross-linking. We probed the conformational changes of the complex during the ATP hydrolysis cycle and unveiled the structural basis of the Mot1–NC2 interplay. Our findings greatly contribute not only to our limited understanding of Mot1 action, but all SNF2/SWI2 family remodeling enzymes.

scholarly journals Crystal structure ofAquifex aeolicusσNbound to promoter DNA and the structure of σN-holoenzyme

2017 ◽  
Vol 114 (10) ◽  
pp. E1805-E1814 ◽  
Author(s):  
Elizabeth A. Campbell ◽  
Shreya Kamath ◽  
Kanagalaghatta R. Rajashankar ◽  
Mengyu Wu ◽  
Seth A. Darst
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Transcription Activity ◽  
X Ray ◽  
Structure And Stability ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Promoter Specificity ◽  
Promoter Dna

The bacterial σ factors confer promoter specificity to the RNA polymerase (RNAP). One alternative σ factor, σN, is unique in its structure and functional mechanism, forming transcriptionally inactive promoter complexes that require activation by specialized AAA+ATPases. We report a 3.4-Å resolution X-ray crystal structure of a σNfragment in complex with its cognate promoter DNA, revealing the molecular details of promoter recognition by σN. The structure allowed us to build and refine an improved σN-holoenzyme model based on previously published 3.8-Å resolution X-ray data. The improved σN-holoenzyme model reveals a conserved interdomain interface within σNthat, when disrupted by mutations, leads to transcription activity without activator intervention (so-called bypass mutants). Thus, the structure and stability of this interdomain interface are crucial for the role of σNin blocking transcription activity and in maintaining the activator sensitivity of σN.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
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 Structural basis of ECF-σ-factor-dependent transcription initiation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Wei Lin ◽  
Sukhendu Mandal ◽  
David Degen ◽  
Min Sung Cho ◽  
Yu Feng ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Structural Basis ◽  
Σ Factor

scholarly journals Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

2003 ◽  
Vol 279 (4) ◽  
pp. 2825-2831 ◽  
Author(s):  
Matthew B. Renfrow ◽  
Nikolai Naryshkin ◽  
L. Michelle Lewis ◽  
Hung-Ta Chen ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Start Site ◽  
Transcription Start ◽  
Initiation Complex ◽  
Factor B ◽  
Transcription Initiation Complex ◽  
Archaeal Transcription ◽  
Promoter Dna
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