The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation

Abstract Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

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Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽

10.7554/elife.70090 ◽

2021 ◽

Vol 10 ◽

Author(s):

Abhishek Mazumder ◽

Richard H Ebright ◽

Achillefs Kapanidis

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Transcription Initiation ◽

Core Promoter ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Extensive Study ◽

Active Centre ◽

Transient Nature ◽

Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4956 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4956-4963 ◽

Cited By ~ 11

Author(s):

X Gong ◽

C A Radebaugh ◽

G K Geiss ◽

M N Simon ◽

M R Paule

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Acanthamoeba Castellanii ◽

Initiation Site ◽

Initiation Factor ◽

Cross Linking ◽

Contour Length ◽

Apparent Homogeneity ◽

Scanning Transmission ◽

Promoter Dna

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

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The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

10.1101/278838 ◽

2018 ◽

Author(s):

Diego Duchi ◽

Abhishek Mazumder ◽

Anssi M. Malinen ◽

Richard H. Ebright ◽

Achillefs N. Kapanidis

Keyword(s):

Rna Polymerase ◽

Active Center ◽

Single Molecule ◽

Transcription Initiation ◽

Stringent Response ◽

Elongation Complex ◽

Conformational States ◽

Closed State ◽

Single Molecule Fret ◽

Promoter Dna

ABSTRACTRNA polymerase (RNAP) contains a mobile structural module, the “clamp,” that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including loading of promoter DNA into the RNAP active-center cleft, unwinding of promoter DNA, transcription elongation complex stability, transcription pausing, and transcription termination. Crystal structures and single-molecule FRET studies establish that the clamp can adopt open and closed conformational states; however, the occurrence, pathway, and kinetics of transitions between clamp states have been unclear. Using single-molecule FRET (smFRET) on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme exists in three distinct conformational states: the previously defined open state, the previously defined closed state, and a previously undefined partly closed state. smFRET time-traces show dynamic transitions between open, partly closed, and closed states on the 0.1-1 second time-scale. Similar analyses of transcription initiation complexes confirm that the RNAP clamp is closed in the catalytically competent transcription initiation complex and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, in contrast to in RNAP holoenzyme, the clamp does not interconvert between the closed state and other states. The stringent-response alarmone ppGpp selectively stabilizes the partly-closed-clamp state, inhibiting interconversion between the partly closed state and the open state. The methods of this report should allow elucidation of clamp conformation and dynamics during all phases of transcription.SIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase “crab-claw” structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements.

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Transcription initiation at a consensus bacterial promoter proceeds via a "bind-unwind-load-and-lock" mechanism

10.1101/2021.03.28.437135 ◽

2021 ◽

Author(s):

Abhishek Mazumder ◽

Richard H Ebright ◽

Achillefs Kapanidis

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Transcription Initiation ◽

Core Promoter ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Extensive Study ◽

Active Centre ◽

Transient Nature ◽

Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, bind-unwind-load-and-lock, model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

10.1101/237123 ◽

2017 ◽

Cited By ~ 1

Author(s):

Wei Lin ◽

Kalyan Das ◽

David Degen ◽

Abhishek Mazumder ◽

Diego Duchi ◽

...

Keyword(s):

Binding Site ◽

Single Molecule ◽

Transcription Initiation ◽

Resonance Energy ◽

Active Pharmaceutical Ingredient ◽

Conformational State ◽

Initiation Factor ◽

Structural Basis ◽

Cross Resistance ◽

Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

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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 of RNA polymerase inhibition by viral and host factors

Nature Communications ◽

10.1038/s41467-021-25666-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Simona Pilotto ◽

Thomas Fouqueau ◽

Natalya Lukoyanova ◽

Carol Sheppard ◽

Soizick Lucas-Staat ◽

...

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Conformational Changes ◽

Transcription Initiation ◽

Environmental Changes ◽

Initiation Factor ◽

Structural Basis ◽

Regulation Of Transcription ◽

Domains Of Life ◽

Sulfolobus Turreted Icosahedral Virus

AbstractRNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

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Structural basis of RNA polymerase inhibition by viral and host factors

10.21203/rs.3.rs-591334/v1 ◽

2021 ◽

Author(s):

Finn Werner ◽

Simona Pilotto ◽

Thomas Fouqueau ◽

Natalya Lukoyanova ◽

Carol Sheppard ◽

...

Keyword(s):

Dna Binding ◽

Conformational Changes ◽

Transcription Initiation ◽

Environmental Changes ◽

Initiation Factor ◽

Structural Basis ◽

Regulation Of Transcription ◽

Transcript Cleavage ◽

Domains Of Life ◽

Sulfolobus Turreted Icosahedral Virus

Abstract The inhibition of RNA polymerases activity plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural basis underlying RNAP inhibition in archaea. The Acidianus two-tailed virus (ATV) encodes the RIP factor that binds to the inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus (STIV) induces the expression of the host factor TFS4, which binds in the RNAP secondary channel similarly to eukaryotic transcript cleavage factors. In contrast to RIP, TFS4 binding allosterically induces a widening of the DNA binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

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RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920747117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 5801-5809 ◽

Cited By ~ 7

Author(s):

Lingting Li ◽

Vadim Molodtsov ◽

Wei Lin ◽

Richard H. Ebright ◽

Yu Zhang

Keyword(s):

Rna Polymerase ◽

Crystal Structures ◽

Active Center ◽

Transcription Initiation ◽

Initiation Factor ◽

Template Strand ◽

Bacterial Transcription ◽

Transcription Initiation Factor ◽

Σ Factor ◽

Nascent Rna

All organisms—bacteria, archaea, and eukaryotes—have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the “σ-finger” of the bacterial transcription initiation factor σ. The structures reveal that—for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5′-triphosphate RNA and 5′-hydroxy RNA—the “σ-finger” is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5′-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.

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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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