Structural Basis for Control of Bacterial RNA Polymerase Pausing by a Riboswitch and its Ligand

Abstract Folding of nascent transcripts can be modulated by the proximal RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (que-ePEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination, however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-ePEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, upon ligand binding the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which small nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

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Structural basis for 2′-deoxyguanosine recognition by the 2′-dG-II class of riboswitches

Nucleic Acids Research ◽

10.1093/nar/gkz839 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10931-10941 ◽

Cited By ~ 3

Author(s):

Michal M Matyjasik ◽

Robert T Batey

Keyword(s):

Ligand Binding ◽

Consensus Sequence ◽

Bioinformatic Analysis ◽

Binding Pocket ◽

Structural Basis ◽

Binding Core ◽

Nucleotide Insertion ◽

Parental Class ◽

Purine Riboswitch ◽

Aptamer Domain

Abstract A recent bioinformatic analysis of well-characterized classes of riboswitches uncovered subgroups unable to bind to the regulatory molecule of the parental class. Within the guanine/adenine class, seven groups of RNAs were identified that deviate from the consensus sequence at one or more of three positions directly involved purine nucleobase recognition, one of which was validated as a second class of 2′-deoxyguanosine riboswitch (called 2′-dG-II). To understand how 2′-dG-II riboswitches recognize their cognate ligand and how they differ from a previously identified class of 2′-deoxyguanosine binding riboswitches, we have solved the crystal structure of a 2′-dG-II aptamer domain bound to 2′-deoxyguanosine. This structure reveals a global architecture similar to other members of the purine riboswitch family, but contains key differences within the ligand binding core. Defining the 2′-dG-II riboswitches is a two-nucleotide insertion in the three-way junction that promotes novel base-base interactions. Unlike 2′-dG-I riboswitches, the 2′-dG-II class only requires local changes to the ligand binding pocket of the guanine/adenine class to achieve a change in ligand preference. Notably, members of the 2′-dG-II family have variable ability to discriminate between 2′-deoxyguanosine and riboguanosine, suggesting that a subset of 2′-dG-II riboswitches may bind either molecule to regulate gene expression.

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RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement

Nature Structural & Molecular Biology ◽

10.1038/nsmb.2867 ◽

2014 ◽

Vol 21 (9) ◽

pp. 794-802 ◽

Cited By ~ 67

Author(s):

Pyae P Hein ◽

Kellie E Kolb ◽

Tricia Windgassen ◽

Michael J Bellecourt ◽

Seth A Darst ◽

...

Keyword(s):

Rna Polymerase ◽

Structure Formation ◽

Rna Structure ◽

Domain Movement ◽

Nascent Rna ◽

Polymerase Pausing

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Structural basis for transcription complex disruption by the Mfd translocase

eLife ◽

10.7554/elife.62117 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jin Young Kang ◽

Eliza Llewellyn ◽

James Chen ◽

Paul Dominic B Olinares ◽

Joshua Brewer ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

Transcription Coupled Repair

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

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Structural basis for transcription complex disruption by the Mfd translocase

10.1101/2020.08.12.248500 ◽

2020 ◽

Author(s):

Jin Young Kang ◽

Eliza Llewellyn ◽

James Chen ◽

Paul Dominic B. Olinares ◽

Joshua Brewer ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

Transcription Coupled Repair

SummaryTranscription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

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Structural Basis for Recognition and Sequestration of UUUOH 3′ Temini of Nascent RNA Polymerase III Transcripts by La, a Rheumatic Disease Autoantigen

Molecular Cell ◽

10.1016/j.molcel.2005.10.027 ◽

2006 ◽

Vol 21 (1) ◽

pp. 75-85 ◽

Cited By ~ 96

Author(s):

Marianna Teplova ◽

Yu-Ren Yuan ◽

Anh Tuân Phan ◽

Lucy Malinina ◽

Serge Ilin ◽

...

Keyword(s):

Rheumatic Disease ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Structural Basis ◽

Polymerase Iii ◽

Nascent Rna

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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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