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  • The EMBO Journal (2002) 21, 1369 - 1378
  • doi:10.1093/emboj/21.6.1369

Structure-based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA–DNA hybrid length

Konstantin Kuznedelov1,2,4, Nataliya Korzheva3,4, Arkady Mustaev3 and Konstantin Severinov1

  1. Waksman Institute, Rutgers, The State University, Piscataway, NJ 08854, USA
  2. Limnological Institute of Siberian Branch of Russian Academy of Sciences, Irkutsk 664033, Russia
  3. Public Health Research Institute, New York, NY 10016, USA
  4. K.Kuznedelov and N.Korzheva contributed equally to this work

Correspondence to:

Konstantin Severinov, E-mail: severik@waksman.rutgers.edu

Received 17 December 2001; Accepted 18 January 2002; Revised 18 January 2002

Analysis of multisubunit RNA polymerase (RNAP) structures revealed several elements that may constitute the enzyme's functional sites. One such element, the 'rudder', is formed by an evolutionarily conserved segment of the largest subunit of RNAP and contacts the nascent RNA at the upstream edge of the RNA–DNA hybrid, where the DNA template strand separates from the RNA transcript and re-anneals with the non-template strand. Thus, the rudder could (i) maintain the correct length of the RNA–DNA hybrid; (ii) stabilize the nascent RNA in the complex; and (iii) promote or maintain localized DNA melting at the upstream edge of the bubble. We generated a recombinant RNAP mutant that lacked the rudder and studied its properties in vitro. Our results demonstrate that the rudder is not required for establishment of the upstream boundary of the transcription bubble during promoter complex formation, nor is it required for separation of the nascent RNA from the DNA template strand or transcription termination. Our results suggest that the rudder makes critical contributions to elongation complex stability through direct interactions with the nascent RNA.

  • Keywords:

    • localized DNA melting,
    • RNA polymerase,
    • transcription complex,
    • transcription complex stability,
    • transcription termination