Manli Jiang ^{1, 2, 4} , Na Ma ^{1, 2, 4}, Dmitry G. Vassylyev  ³, and William T. McAllister ¹

¹ Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 USA
² Graduate Program in Molecular and Cellular Biology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 USA
³ Structurome Research Group and Cellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, 1-1-2 Hyogo 679-5148, Japan
⁴ These authors contributed equally to this work.

Correspondence: William T. McAllister;  (718) 270-1331 (phone);   (718) 270-2656 (fax)
william.mcallister@downstate.edu

Abstract:

Unlike DNA polymerases, RNA polymerases (RNAPs) must displace the nascent product from the template
and restore the DNA to duplex form after passage of the transcription complex. To accomplish this, RNAPs
establish a locally denatured “bubble” that encloses a short RNA:DNA hybrid. As the polymerase advances
along the template, the RNA is displaced at the trailing edge of the bubble and the two DNA strands are
reannealed. Structural analyses have revealed a number of elements that are likely to be involved in this process in T7 RNAP. In this work, we used genetic and biochemical methods to explore the roles of these elements during the transition from an initiation complex to an elongation complex. The results indicate that the transition is a multistep process and reveal a critical role for the nontemplate strand of the DNA.

Figure 1. Formation of the RNA Exit Pore during the Transition to an EC (elongation complex) .

Peptides that are involved in the crosslink to the RNA nucleotide at -14 (723–743, 751–783, 290–315) and at -9 (744–750), as well as other important regions, are mapped onto the structure of T7 RNAP in the IC (initiation complex, upper panels) and EC (elongation complex,  lower panels) (Cheetham and Steitz 1999; Tahirov et al. 2002 and Yin and Steitz 2002). The T (template) and NT (nontemplate) strands of the DNA are in red and blue, respectively. The view is into the RNA exit pore; the displaced RNA (yellow) is observed emerging toward the viewer (lower panel). The side chains of residues K302 and K303 are in ball-and-stick representation. A surface view indicating electrostatic potential (positive charge, blue; negative, red; neutral, white) is shown in the right portion of each panel (same orientation).
 

Figure 6. Organization of the Upstream and Downstream Edges of the Transcription Bubble.

(A) The upstream boundary of the transcription bubble (Tahirov et al. 2002 and Yin and Steitz 2002).
         DNA and RNA are colored as in Figure 1. The view of the transcription complex relative to that of
         Figure 1 has been rotated ~90° around the vertical axis such that the displaced RNA is directed
         toward the left rear of the viewing plane. The RNA displacement loop (residues 56–71) is green,
         and the side chains of Q58, E63, and D66 are highlighted. The flap domain (residues 152–204) is in
         magenta, and the side chains of K172 and R173 are highlighted. The tip of the specificity loop
         (residues 749–753) is in cyan. A portion of the thumb domain (residues 384–402) that makes
         contacts with the RNA:DNA hybrid is in orange; the side chain of Y385 (substitution of which
         results in failure to terminate at a class II termination signal [Brieba et al., 2000]) is highlighted.

(B)
         The downstream edge of the bubble. The complex has been rotated such that the displaced RNA
         exits to the rear and upper right of the viewing plane. Note that upstream elements of the flap
         domain (magenta; K163, K164) approach closely to the point of separation of the T (template) and NT
         (nontemplate) strands at the leading edge of the bubble in a similar manner as K172 and R173 at the
         trailing edge of the bubble (left panel). The intervening portion of the flap domain forms part of the NT
         strand binding channel.

Is thermodynamics the ultimate arbiter of chemical reactions ? Only in closed systems at equilibrium. In life, with
a constant flow of ATP mediating unlikely reactions, literally anything is possible if it enhances survival. And yet,
thermodynamics must be considered. The melting temperature of a DNA-DNA helix is lower than that of a
DNA-RNA helix, which, in turn, is lower than that of a RNA-RNA helix. So, double-stranded RNA, even if only
partially helical, must be respected on thermodynamic grounds, if no other.

This new study by Manli Jiang, Na Ma, Dmitry Vassylyev, and William McAllister on the origins and the mechanics of the transcription bubble indicate that the anti-template DNA strand of the gene being transcribed may be almost as important as the template DNA strand, and that product RNA, and perhaps transactive RNA, are playing important roles in selective gene transcription.

Additional References:

1. NetworkEditor: "RNA forms  the Transcription Bubble", in: "RNA and Biological Causality".

2. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription".
 

Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

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