Switching Promotor Recognition of Phage RNA Polymerase in Silico Following Path along Lab Directed Evolution

In this work we computationally investigated how a viral RNA polymerase (RNAP) from bacteriophage T7 evolves into RNAP variants under lab-directed evolution to switch recognition from T7 promoter to T3 promoter in transcription initiation. We first constructed a closed initiation complex for the wild-type T7 RNAP, and then for six mutant RNAPs discovered from phage assisted continuous evolution experiments. All-atom molecular dynamics (MD) simulations up to one microsecond each were conducted on these RNAPs in complex with T7/T3 promoter. Our simulations show notably that protein-DNA electrostatic interactions or stabilities at the RNAP-DNA promoter interface well dictate the promoter recognition preference of the RNAP and variants. Key residues and structural elements that contribute significantly to switching the promoter recognition were identified. Followed by a first point mutation N748D on the specificity loop to slightly disengage the RNAP from the promoter to hinder the original recognition, we found an auxiliary helix (206-225) that takes over switching the promoter recognition upon further mutations (E222K and E207K), by forming additional charge interactions with the promoter DNA and reorientating differently on the T7 and T3 promoter. Further mutations on the AT-rich loop and the specificity loop can fully switch the RNAP-promoter recognition to the T3 promoter. Overall, our studies reveal energetics and structural dynamics details along an exemplary directed evolutionary path of the phage RNAP variants for a rewired promoter recognition function. The findings demonstrate underlying physical mechanisms and are expected to assist knowledge/data learning or rational redesign of the protein enzyme structure-function.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520555113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4051-4056 ◽

Cited By ~ 54

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

Acta Biochimica Polonica ◽

10.18388/abp.2003_3623 ◽

2003 ◽

Vol 50 (4) ◽

pp. 909-920 ◽

Cited By ~ 4

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.

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Direct binding of TFEα opens DNA binding cleft of RNA polymerase

Nature Communications ◽

10.1038/s41467-020-19998-x ◽

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.

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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 ◽

10.1021/bi971432y ◽

1997 ◽

Vol 36 (45) ◽

pp. 13954-13962 ◽

Cited By ~ 20

Author(s):

Amarendra Kumar ◽

Smita S. Patel

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Ternary Complex ◽

T7 Rna Polymerase ◽

Promoter Dna

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Bacterial RNA polymerase can retain σ70 throughout transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513899113 ◽

2016 ◽

Vol 113 (3) ◽

pp. 602-607 ◽

Cited By ~ 35

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.

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E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

10.1101/766725 ◽

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910827116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 18923-18927 ◽

Cited By ~ 5

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.

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Investigations of the modular structure of bacterial promoters

Biochemical Society Symposium ◽

10.1042/bss0730001 ◽

2006 ◽

Vol 73 ◽

pp. 1-10 ◽

Cited By ~ 30

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.

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Architecture of Bacterial Promoters; The case of the Escherichia coli ogt Promoter

Journal of Molecular Biology Research ◽

10.5539/jmbr.v6n1p111 ◽

2016 ◽

Vol 6 (1) ◽

pp. 111 ◽

Cited By ~ 1

Author(s):

Aida Ansarikaleibari

Keyword(s):

Rna Polymerase ◽

Binding Site ◽

Transcription Initiation ◽

Molecular Model ◽

Point Mutations ◽

Class Ii ◽

Transcription Activator ◽

Promoter Recognition ◽

Opposite Face ◽

Domain 4

All bacteria utilize RNA polymerase enzyme and transcription activator proteins to regulate gene expression in response to internal or external stress. Some bacterial promoters are regulated with only one transcription factor whilst two or more transcription activators regulate some other promoters. NarL is a transcription activator protein that activates the E. coli yeaR and ogt promoters in response to nitrate and nitrite induction in absence of oxygen. In the present study we have studied ogt1052 promoter, which is a derivative of ogt promoter containing only one NarL binding site very close to -35 element. Therefore, it is considered as class II activator dependent promoter just as yeaR promoter. A molecular structure of ogt1052 promoter was proposed which suggests that NarL binding site is located in opposite face of DNA that contains Alpha-CTD and sigma domain 4 of RNA polymerase enzyme required for promoter recognition. The aim of the present study was to study and test the suggested molecular model by creating point mutations at -35 element and deletion of one base pair in spacer region, to test whether sigma domain 4 is necessary to bind -35 hexamer in order to start transcription initiation, and to test whether NarL activates the promoter by interaction with Alpha-CTD in the opposite face of the DNA. Based on the result achieved, ogt1052 promoter is a class I promoter “dressed” like a class II promoter.

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