Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: Crystal structures of transcription initiation complexes engaged in standard transcription initiation and reiterative transcription initiation. Left, experimental electron density (mFo-DFc; contoured at 2.0σ in A and 1.5σ in B-D) and atomic model, showing interactions of RNAP and σ with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA (RNAP β subunit and β’ non-conserved domain omitted for clarity). Right, nucleic-acid scaffold. RNAP, gray; RNAP active-center catalytic Mg 2+ (I) ion, violet filled circle; σ, yellow; σ finger, asterisk (left subpanel) and yellow-brown (right subpanel); σR3-σR4 linker in RNA exit channel, brown; −10 element of DNA nontemplate strand, dark blue; discriminator element of DNA nontemplate strand, light blue; rest of DNA nontemplate strand, pink; DNA template strand, red; RNA product, magenta. Cyan rectangles on panel B indicate disordered regions containing scrunched nucleotides. Cyan rectangles in panels C and D indicate ordered scrunched nucleotides. Bulged-out nucleotides in panels B-D, right, indicate bulged-out scrunched nucleotides. Violet rectangles indicate RNA-DNA hybrids. Raised template-strand nucleotides in panels C-D indicate non-base-paired nucleotides. (A) RNAP-promoter open complex (RPo; PDB 4G7H; ). (B) RNAP-promoter initial transcribing complex containing 5 nt RNA product generated by in crystallo standard transcription initiation (RPitc,5). (C) RNAP-promoter reiteratively transcribing complex containing 5 nt RNA product generated by in crystallo reiterative transcription initiation on nucleic-acid scaffold having a template-strand G +1 G +2 G +3 homopolymeric sequence (RPrtc,5 [G +1 G +2 G +3 ]). (D) RNAP-promoter reiteratively transcribing complex containing 4 nt RNA product generated by in crystallo reiterative transcription initiation on nucleic-acid scaffold having a template-strand C +1 C +2 C +3 homopolymeric sequence (RPrtc,4 [C +1 C +2 C +3 ]).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Generated, Sequencing

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A) RNA-DNA base pairing in crystal structures of transcription initiation complexes engaged in standard transcription initiation (RPitc,5) and reiterative transcription initiation (RPrtc,5 [G +1 G +2 G +3 ] and RPrtc,4 [C +1 C +2 C +3 ]). Left, template-strand DNA bases (red) and corresponding RNA bases (magenta) in view orientation parallel to RNA-DNA-hybrid helix axis. Right, template-strand DNA bases (red) and corresponding RNA bases (magenta) in view orientation perpendicular to RNA-DNA-hybrid helix axis. Positions are numbered relative to the RNAP active-center P site. Dashed lines indicate Watson-Crick H-bonds. Violet rectangles indicate RNA-DNA hybrids. At positions P-4, P-3, and P-2 of RPrtc,5 [G +1 G +2 G +3 ], and at positions P-3 and P-2 of RPrtc,4 [C +1 C +2 C +3 ], template-strand DNA bases are displaced relative to their locations in RPitc,5, and no base pairing occurs. (B) Superimposition of DNA template strand and RNA of RPrtc,5 [G +1 G +2 G +3 ] (red spheres, DNA phosphates; magenta spheres, RNA phosphates; violet sphere, RNAP active-center catalytic Mg 2+ ion) on DNA template strand and RNA of RPitc,5 (gray spheres, DNA and RNA phosphates). Left, view orientation parallel to RNA-DNA-hybrid helix axis; right, view orientation perpendicular to RNA-DNA-hybrid helix axis. Distances in cyan, displacement of template-strand DNA nucleotides at positions P-4 and P-3 of RPrtc,5 [G +1 G +2 G +3 ] relative to their locations in RPitc,5.

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques:

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A) In vitro transcription experiments with nucleic-acid scaffolds used in crystal structure determination of - (“partial bubble”) and cryo-EM structure determination of - (“full bubble”). Left , nucleic-acid scaffolds (promoter −35 and −10 elements, blue; promoter discriminator elements, light blue; template-strand homopolymeric sequences, red; non-complementary sequences, raised and lowered nucleotides; transcription start sites, +1). Right , in vitro transcription products with T. thermophilus RNAP σ A holoenzyme ( Tth ) and E. coli RNAP σ 70 holoenzyme ( Eco ) for partial-bubble scaffold 1 (template-strand non-homopolymeric transcription-start-site sequence T +1 G +1 A +1 ; transcription with ATP and UTP; standard transcription initiation), partial-bubble scaffold 2 (template-strand homopolymeric sequence G +1 G +2 G +3 ; transcription with CTP; reiterative transcription initiation), partial-bubble scaffold 3 (template-strand homopolymeric sequence C +1 C +2 C +3 ; transcription with GTP; reiterative transcription initiation), and full-bubble scaffold 4 (template-strand homopolymeric sequence C +1 C +2 C +3 ; transcription with GTP; reiterative transcription initiation). (B) In vitro transcription experiments with bacteriophage T5 N25 promoter derivatives used in photocrosslinking and single-molecule DNA-nanomanipulation experiments of - . Left , promoter sequences (colors as in panel A). Right , in vitro transcription products with and E. coli RNAP σ 70 holoenzyme ( Eco ) for bacteriophage N25 WT (transcription with ATP and UTP; standard transcription initiation), N25 [G +1 G +2 G +3 ] (template-strand homopolymeric sequence G +1 G +2 G +3 ; transcription with CTP; reiterative transcription initiation), and N25 [C +1 C +2 C +3 ] (template-strand homopolymeric sequence C +1 C +2 C +3 ; transcription with GTP; reiterative transcription initiation). RNA products 2-10 nt in length are products of standard transcription initiation or reiterative transcription initiation; RNA products >10 nt in length are products of reiterative transcription initiation.

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: In Vitro, Cryo-EM Sample Prep, Sequencing

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A) Overall structure ( β’ nonconserved region omitted for clarity; two orthogonal view orientations). Dark blue brackets indicate the standard RNA exit and alternative RNA exit. Cyan rectangles indicate scrunched nucleotides. Violet rectangles indicate RNA-DNA hybrids. Other symbols and colors in panels A-D are as in . (B) Left, cryo-EM density and atomic model, showing interactions of RNAP and σ with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA. Right, nucleic-acid scaffold. Yellow-brown, σ finger (note displacement of σ-finger tip); magenta dots, RNA outside RNAP active-center cleft (nucleotides rN≥11). (C) Superimposition of DNA in RPrtc,≥11 [C +1 C +2 C +3 ] (pink and red) on DNA in RPo (black; PDB 512D; ). (D) Close-up of cryo-EM density and atomic model for RNA (nucleotides rN1-rN11 numbered in white).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A) RNA-DNA base pairing. Yellow dashed lines, non-Watson-Crick H-bonds. Other symbols and colors as in . (B) Superimposition of DNA template strand and RNA of RPrtc,≥11 [C +1 C +2 C +3 ] (red spheres, DNA phosphates; magenta spheres, RNA phosphates; violet sphere, RNAP active-center catalytic Mg 2+ ion) on DNA template strand and RNA of RPitc,5 (gray spheres, DNA and RNA phosphates). (C) Protein-RNA and DNA-RNA interactions in alternative RNA path. Left, center, and right subpanels show interactions with RNA nucleotides with rN3-rN6, rN6-rN8, and rN9-rN11 (atoms in magenta; nucleotide numbers in white). Ribbons and sticks, backbone segments and sidechain atoms of residues of RNAP β (backbone segments in white; carbon oxygen and nitrogen atoms in white, red, and blue) and σ (backbone segments in yellow; carbon oxygen and nitrogen atoms in yellow, red, and blue) that make protein-RNA interactions with rN3-rN11. “NT+2,” nucleotide at nontemplate-strand position +2 that makes base-stacking interaction with RNA nucleotide rN8. Other colors as in . (D) σ-finger conformation. Left, cryo-EM density and atomic model for σ finger. Right, superimposition of σ finger of RPrtc,≥11 [C +1 C +2 C +3 ] (yellow) on σ finger of RPo (black; PDB 4G7H; ). Other colors as in . (E) RNAP clamp conformation. Superimposition of RNAP of RPrtc,≥11 [C +1 C +2 C +3 ] (clamp in red; rest of RNAP in gray) on RNAP of RPo (black; PDB 4G7H; ).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: Locations of residues that make protein-RNA interactions with rN6-rN11 in the structure of RPrtc,≥11 [C +1 C +2 C +3 ] in the sequences of RNAP β subunit (left) and σ (right). Sequence alignments for β and σ 70 /σ A for Gram-negative, Gram-positive, and Thermus - Deinococcus -clade bacterial species showing locations of RNAP residues that contact protein-RNA interactions with rN6-rN11 (black rectangles; numbered as in T. thermophilus and, in parentheses, as in E. coli ; identities from ), and locations of RNAP and σ structural elements and conserved regions (black bars; ; ). Species are as follows: E. coli (ECOLI), Salmonella typhimurium (SALTY), Klebsiella pneumoniae (KLEP7), Enterobacter cloacae (ENTCC), Vibrio cholerae (VIBCH), Haemophilus influenzae (HAEIN), Neisseria gonorrhoeae (NEIG1), Stenotrophomonas maltophilia (STPMP), Moraxella catarrhalis (MORCA), Acinetobacter baumannii (ACIBC), Pseudomonas aeruginosa (PSEAE), Staphylococcus aureus (STAAU), Staphylococcus epidermidis (STAEQ), Enterococcus faecalis (ENTFA), Streptococcus pyogenes (STRP1), Streptococcus pneumoniae (STRP2), Clostridium difficile (CDIFF), Mycobacterium tuberculosis (MYCTU), Mycobacterium avium (MYCA1), Mycobacterium abscessus (MYCA9), Thermus aquaticus (THEAQ), and Thermus thermophilus (TTHER), and Deinococcus radiodurans (DEIRA).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A) RNAP trailing-edge and leading-edge positions in transcription initiation complexes at the N25 promoter (WT) and derivatives of the N25 promoter containing template-strand G +1 G +2 G +3 and C +1 C +2 C +3 homopolymeric sequences (G +1 G +2 G +3 and C +1 C +2 C +3 ). First bracketed subpanel, protein-DNA photocrosslinking data for RNAP-promoter open complex (RPo); second bracketed subpanel, protein-DNA photocrosslinking data for transcription initiation complexes engaged in standard transcription initiation (RPitc) and reiterative transcription initiation (RPrtc); third bracketed subpanel, interpretation of protein-DNA photocrosslinking data for RPo; fourth bracketed subpanel, interpretation of protein-DNA photocrosslinking data for RPitc and RPrtc. In third and fourth subpanels, promoter sequences are shown with positions numbered relative to the transcription start site, and with positions of the −10-element, the discriminator element, and the homopolymeric sequence highlighted in blue, light blue, and red. Thick and thin light green bars indicate strong and weak RNAP-trailing-edge crosslinks, and thick and thin dark green bars indicate strong and weak RNAP-leading-edge crosslinks. Bottom of panel, observed modal trailing-edge/leading-edge distances (modal TE-LE distance) and differences in modal TE-LE distance relative to modal TE-LE distance in RPo at wild-type N25 promoter [Δ(modal TE-LE distance)]. (B) Mechanistic interpretation of data in panel A. Three states are shown: RPo, RPitc [specifically, RPitc having a 5 nt RNA product in a post-translocated state (RPitc,5 post), corresponding to the major crosslink in panel A], and RPrtc. Gray, RNAP; yellow, σ; yellow-brown, σ finger (note displacement of σ-finger tip in RPrtc); brown, σ region-3/region-4 linker; light green, trailing-edge Bpa and crosslinking site for trailing-edge Bpa; dark green, leading-edge Bpa and crosslinking site for leading-edge Bpa; black boxes with blue fill, −10-element nucleotides; black boxes with light blue fill, discriminator-element nucleotides; black boxes with red fill, template-strand homopolymeric-sequence nucleotides; other black boxes, other DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides); magenta boxes, RNA nucleotides; violet rectangles, RNA-DNA hybrids; P and A, RNAP active-center product and addition sites. Raised template-strand nucleotides and black x’s indicate non-base-paired nucleotides. Scrunching of nontemplate and template DNA strands is indicated by bulged-out nucleotides. Initial-product formation in both standard transcription initiation and reiterative transcription initiation involves one step of DNA scrunching. RNA extension in standard transcription initiation involves additional DNA scrunching, but RNA extension in reiterative transcription does not.

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: (A)-(B) Experimental approach ( ; ; ). (A), apparatus. (B), end-to-end extension ( l ) of a mechanically stretched, positively supercoiled (top), or negatively supercoiled (bottom), DNA molecule is monitored. Unwinding of n turns of DNA by RNAP results in compensatory gain of n positive supercoils or loss of n negative supercoils, and movement of the bead by n*56 nm. (C) Single-molecule time traces for RPo and RPitc at the N25 promoter (WT; left), and for RPo and RPrtc at derivatives of the N25 promoter containing template-strand G +1 G +2 G +3 and C +1 C +2 C +3 homopolymeric sequences (G +1 G +2 G +3 and C +1 C +2 C +3 ; middle and right). Upper subpanels, positively supercoiled DNA; lower subpanels, negatively supercoiled DNA. Green points, raw data (30 frames/s); red points, averaged data (1 s window); horizontal black lines, unbound and RPo states; dashed horizontal black lines, RPitc and RPrtc states (with the difference in Δ l obs between RPo and RPitc being substantially greater than the difference in Δ l obs between RPo and RPrtc). (D) Single-molecule transition-amplitude histograms for RPo and RPitc at the N25 promoter (WT; left), and for RPo and RPrtc at derivatives of the N25 promoter containing template-strand G +1 G +2 G +3 and C +1 C +2 C +3 homopolymeric sequences (G +1 G +2 G +3 and C +1 C +2 C +3 ; middle and right). Upper subpanels, positively supercoiled DNA; lower subpanels, negatively supercoiled DNA. Vertical dashed lines, means; Δ l obs,pos, transition amplitudes with positively supercoiled DNA; Δ l obs,neg, transition amplitudes with negatively supercoiled DNA. (E) Differences in Δ l obs,pos and DNA unwinding relative to those in RPo at wild-type N25 promoter (means ± 2SEM).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques:

Journal: bioRxiv

Article Title: Structural and mechanistic basis of reiterative transcription initiation

doi: 10.1101/2021.05.08.443277

Figure Lengend Snippet: The part of the σ finger that occupies the RNAP hybrid binding site is colored dark yellow; the part of the σ region-3/region-4 linker that occupies the RNAP RNA-exit channel is colored brown. Non-templated, reiterative-transcription-dependent RNA nucleotides are colored magenta; templated, standard-transcription-dependent RNA nucleotides are colored orange. Other symbols and colors are as in and . (Left) Hypothesized mechanism for synthesis of long RNA products comprising only non-templated, reiterative-transcription-dependent nucleotides (“non-productive” reiterative transcription initiation by RPrtc, without displacement of the σ finger, displacement of the σ region-3/region-4 linker, and promoter escape). (Right) Hypothesized mechanism for synthesis of long RNA products comprising non-templated, reiterative-transcription-dependent nucleotides at their 5’ ends followed by templated, standard-transcription-dependent nucleotides (“productive” reiterative transcription initiation, entailing switching from reiterative transcription initiation by RPitc to standard transcription initiation by RPrtc → itc, resulting in displacement of the σ finger, displacement of the σ region-3/region-4 linker, promoter escape, optional σ release, and transcription elongation using an RNAP-stepping mechanism).

Article Snippet: Bpa-containing E. coli RNAP core enzyme derivatives RNAP - β’ R1148Bpa and RNAP - β’ T48Bpa were prepared from E. coli strain NiCo21(DE3) (New England Biolabs, Inc.) transformed with plasmid pEVOL-pBpF ( ) and either plasmid pIA900-RNAP - β’ R1148Bpa ( ) or plasmid pIA900-RNAP - β’ T48Bpa , using procedures as in ( ).

Techniques: Binding Assay

Journal: Medical Journal, Armed Forces India

Article Title: Hepatitis G Virus: Prevalence in Blood Donors in Armed Forces

doi: 10.1016/S0377-1237(05)80057-7

Figure Lengend Snippet: Reverse transcriptase polymerase chain reaction confirming Hepatitis G virus

Article Snippet: Briefly, all serum samples were tested for the presence of HGV RNA in duplicate by nested RT-PCR. cDNA synthesis was carried out at 42°C for one hour using external antisense primers and reverse transcriptase enzyme (Bangalore Genei) using a thermal cycler (Lab System, Finland).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Virus