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RNA Extension Drives a Stepwise Displacement of an Initiation-Factor Structural Module in Initial Transcription

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RNA Extension Drives a Stepwise Displacement of an Initiation-Factor Structural Module in Initial Transcription RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription Lingting Lia,b,1, Vadim Molodtsovc,d,1, Wei Linc, Richard H. Ebrightc,d,2, and Yu Zhanga,c,d,2 aKey Laboratory of Synthetic Biology, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032 Shanghai, China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; and cWaksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854; and dDepartment of Chemistry, Rutgers University, Piscataway, NJ 08854 Edited by Lucia B. Rothman-Denes, The University of Chicago, Chicago, IL, and approved February 7, 2020 (received for review November 25, 2019) All organisms—bacteria, archaea, and eukaryotes—have a tran- and CSB, the Rrn7 zinc ribbon and B-reader, the TFIIB zinc scription initiation factor that contains a structural module that ribbon and B-reader, and the Brf1 zinc ribbon, respectively, all of binds within the RNA polymerase (RNAP) active-center cleft and which are structurally related to each other but are structurally interacts with template-strand single-stranded DNA (ssDNA) in the unrelated to the bacterial σ-finger and σ54 RII.3 (16–18, 21–27). immediate vicinity of the RNAP active center. This transcription It has been apparent for nearly two decades that the relevant initiation-factor structural module preorganizes template-strand structural module must be displaced before or during initial ssDNA to engage the RNAP active center, thereby facilitating bind- transcription (1–3, 5–12, 17, 18, 28–33). Changes in protein-DNA ing of initiating nucleotides and enabling transcription initiation photo-crosslinking suggestive of displacement have been reported from initiating mononucleotides. However, this transcription (1), and changes in profiles of abortive initiation and initial- initiation-factor structural module occupies the path of nascent transcription pausing upon mutation of the relevant structural RNA and thus presumably must be displaced before or during – initial transcription. Here, we report four sets of crystal structures module, have been reported (1, 9, 24, 28, 29, 31 33, 35, 36). of bacterial initially transcribing complexes that demonstrate and However, the displacement has not been observed directly. As a define details of stepwise, RNA-extension-driven displacement of result, it has remained unclear whether the displacement occurs in BIOCHEMISTRY the “σ-finger” of the bacterial transcription initiation factor σ. The asinglesteporinmultiplesteps, where the displaced residues structures reveal that—for both the primary σ-factor and extra- move, and what drives the displacement. cytoplasmic (ECF) σ-factors, and for both 5′-triphosphate RNA and Here, we present four sets of crystal structures of bacterial initially 5′-hydroxy RNA—the “σ-finger” is displaced in stepwise fashion, transcribing complexes that demonstrate—and define structural and progressively folding back upon itself, driven by collision with the RNA 5′-end, upon extension of nascent RNA from ∼5ntto∼10 nt. Significance transcription initiation | initial transcription | promoter escape | sigma The “σ-finger” of bacterial initiation factor σ binds within RNA factor | initiation factor polymerase active-center cleft and blocks the path of nascent RNA. It has been hypothesized that the σ-finger must be dis- ll cellular RNA polymerases (RNAPs)—bacterial, archaeal, placed during initial transcription. By determining crystal struc- Aand eukaryotic—require a transcription initiation factor, or tures defining successive steps in initial transcription, we a set of transcription initiation factors, to perform promoter- demonstrate that the σ-finger is displaced in a stepwise fashion, specific transcription initiation (1–4). In all promoter-specific tran- driven by collision with the RNA 5′-end, as nascent RNA is ex- scription initiation complexes—bacterial, archaeal, and eukaryotic—a tended from ∼5ntto∼10 nt during initial transcription. We structural module of a transcription initiation factor enters the further show that this is true for both the primary σ-factor and RNAP active-center cleft and interacts with template-strand alternative σ-factors, and that the nascent RNA length at which single-stranded DNA (ssDNA) close to the RNAP active center displacement occurs is determined by the extent to which the (2, 5–27). Deletion of the structural module results in severely σ-finger penetrates the active center of RNA polymerase. impaired transcription activity (6–9, 28, 29). This transcription initiation-factor structural module preorganizes template-strand Author contributions: R.H.E. and Y.Z. designed research; L.L., V.M., W.L., and Y.Z. per- ssDNA to adopt a helical conformation and to engage the formed research; L.L., V.M., R.H.E., and Y.Z. analyzed data; and R.H.E. and Y.Z. wrote the paper. RNAP active center, thereby facilitating binding of initiating nu- The authors declare no competing interest. cleotides and enabling primer-independent transcription initiation from initiating mononucleotides (1–9, 11, 12, 16–19, 24, 27–29). This article is a PNAS Direct Submission. However, this transcription initiation-factor structural module Published under the PNAS license. occupies the path that will be occupied by nascent RNA during Data deposition: Coordinates have been deposited in the Protein Data Bank (PDB) under ID codes 6LTS, 6KQD, 6KQE, 6KQF, 6KQG, and 6KQH for Tt σA(△’513’-’519’)-RPo, Tt σA- initial transcription and thus presumably must be displaced before RPitc3, Tt σA-RPitc4, Tt σA-RPitc5, Tt σA-RPitc6, and Tt σA-RPitc7, respectively; coordinates or during initial transcription (1–3, 5–12, 17, 18, 28, 30–33). have been deposited in the PDB under ID codes 6L74, 6KQL, 6KQM, and 6KQN for Tt σA- In bacterial transcription initiation complexes containing the RPitc2-PPP, Tt σA-RPitc4-PPP, Tt σA-RPitc5-PPP, and Tt σA-RPitc6-PPP, respectively; coordi- primary σ-factor and most alternative σ-factors, the relevant nates have been deposited in the PDB under ID codes 6KON, 6KOO, 6KOP, and 6KOQ for σ “σ ” Mtb σH-RPitc5, Mtb σH-RPitc7, Mtb σH-RPitc9, and Mtb σH-RPitc10, respectively; and structural module is the -factor -finger (also referred to as coordinates have been deposited in the PDB under ID codes 6TYE, 6TYF, and 6TYG for σR3.2, σR3-σR4 linker, or the σR2-σR4 linker) (1, 2, 5–10, 14, Mtb σL-RPitc5, Mtb σL-RPitc6, and Mtb σL-RPitc9, respectively. 34). In bacterial transcription initiation complexes containing the 1L.L. and V.M. contributed equally to this work. σ σ54 σ54 alternative -factor, , the relevant structural module is the 2To whom correspondence may be addressed. Email: [email protected] or RII.3 region, which is structurally unrelated to the σ-finger (13, 15). [email protected]. In archaeal RNAP-, eukaryotic RNAP-I-, eukaryotic RNAP-II-, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and eukaryotic RNAP-III-dependent transcription initiation com- doi:10.1073/pnas.1920747117/-/DCSupplemental. plexes, the relevant structural modules are the TFB zinc ribbon www.pnas.org/cgi/doi/10.1073/pnas.1920747117 PNAS Latest Articles | 1of9 Downloaded by guest on September 26, 2021 — σ mechanistic details of displacement of the -finger during initial template A finger transcription. The first and second sets are structures of initial A strand transcribing complexes comprising Thermus thermophilus (Tth) RNAP, the Tth primary σ-factor, σA,promoterDNA,andeither 5′-hydroxyl-containing RNAs 2, 3, 4, 5, 6, and 7 nt in length (σA- RPitc2, σA-RPitc3, σA-RPitc4, σA-RPitc5, σA-RPitc6, and σA- RPitc7; Fig. 1) or comprising Tth RNAP, Tth σA,promoterDNA, ′ Mg2+ and 5 -triphosphate-containing RNAs, 2, 4, 5, and 6 nt in length σA-RPo (σA-RPitc2-PPP, σA-RPitc4-PPP, σA-RPitc5-PPP, and σA-RPitc6- template A finger PPP; Fig. 2). These sets correspond to, respectively, six successive B strand intermediates in primary σ-factor-dependent, primer-dependent transcription initiation (5′-hydroxyl-containing RNA; ref. 2), and σ four successive intermediates in primary -factor-dependent, primer- +2 independent transcription initiation (5-triphosphate-containing RNA; ref. 2). The third and fourth sets are structures of initial RNA 5’ +1 Mycobacterium tuberculosis A end -1 Mg2+ transcribing complexes comprising σ -RPitc2 2-nt RNA -2 (Mtb)RNAP,Mtb extracytoplasmic-function (ECF) σ-factor σH, promoter DNA, and 5′-hydroxyl-containing RNA oligomers 5, 6, C template A finger 7, 9, and 10 nt in length (σH-RPitc5, σH-RPitc6, σH-RPitc7, σH- strand RPitc9, and σH-RPitc10; Fig. 3) and comprising Mtb RNAP, Mtb σ σL ′ ECF -factor , promoter DNA, and 5 -hydroxyl-containing +2 RNA oligomers 5, 6, and 9 nt in length (σL-RPitc5, σL-RPitc6, σL and -RPitc9; Fig. 4). These sets correspond to, respectively, five RNA 5’ +1 intermediates in alternative σ-factor-dependent transcription ini- end -1 Mg2+ -2 tiation with ECF σ-factor σH, and three intermediates in alterna- 3-nt RNA σ σ tive -factor-dependent transcription initiation with ECF -factor template A finger σL. Taken together, the structures establish that—for both the D strand primary σ-factor and ECF σ-factors, and for both 5′-hydroxyl RNA ′ — σ and 5 -triphosphate RNA the -finger is displaced in stepwise RNA 5’ fashion, progressively folding back upon itself, driven by colli- end +2 sion with the RNA 5′-end, upon extension of nascent RNA from ∼ ∼ +1 5ntto 10 nt. -1 2+ -3 -2 Mg σA-RPitc4 4-nt RNA Results template A finger Stepwise, RNA-Driven Displacement of the σ-Finger: Primary σ,5′- E strand Hydroxyl RNA. The primary σ-factor, responsible for transcription σA σ70 initiation at most promoters under most conditions, is ( in RNA 5’ Escherichia coli; ref. 1). The σ-finger of σA (residues 507 to 520; end +2 σA residues numbered as in E. coli σ70 here and hereafter) con- tacts template-strand ssDNA nucleotides at positions −4to−3 -4 +1 -1 2+ and preorganizes template-strand ssDNA to adopt a helical con- -3 -2 Mg σA-RPitc5 5-nt RNA formation and to engage the RNAP active center (5, 11, 12).
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