Inhibition of T7 RNA Polymerase:  Transcription Initiation and Transition from Initiation to Elongation Are Inhibited by T7 Lysozymeviaa Ternary Complex with RNA Polymerase and Promoter DNA†

Biochemistry ◽  
1997 ◽  
Vol 36 (45) ◽  
pp. 13954-13962 ◽  
Author(s):  
Amarendra Kumar ◽  
Smita S. Patel
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Ternary Complex ◽  
T7 Rna Polymerase ◽  
Promoter Dna

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 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
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 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 E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

2019 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y. Chen ◽  
Elizabeth A. Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
E Coli ◽  
Bacterial Proteins ◽  
Cryo Electron Microscopy ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

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.

Investigations of the modular structure of bacterial promoters

2006 ◽  
Vol 73 ◽  
pp. 1-10 ◽  
Author(s):  
Nora S. Miroslavova ◽  
Stephen J.W. Busby
Keyword(s):  
Rna Polymerase ◽  
Dna Sequence ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Modular Structure ◽  
Bacterial Rna ◽  
Sequence Elements ◽  
Promoter Dna ◽  
Transcript Initiation ◽  
Rna Polymerase Holoenzyme

Bacterial RNA polymerase holoenzyme carries different determinants that contact different promoter DNA sequence elements. These contacts are essential for the recognition of promoters prior to transcript initiation. Here, we have investigated how active promoters can be built from different combinations of elements. Our results show that the contribution of different contacts to promoter activity is critically dependent on the overall promoter context, and that certain combinations of contacts can hinder transcription initiation.

scholarly journals Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

2016 ◽  
Vol 113 (15) ◽  
pp. 4051-4056 ◽  
Author(s):  
Bin Liu ◽  
Yuhong Zuo ◽  
Thomas A. Steitz
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Active Site ◽  
Transcription Initiation ◽  
De Novo ◽  
Addition Reaction ◽  
Specific Interactions ◽  
Promoter Recognition ◽  
Nucleotide Addition ◽  
Promoter Dna

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

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