HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 41, 30581-30592, October 13, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/41/30581    most recent M605209200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Goede, B. Articles by Thomm, M. Search for Related Content PubMed PubMed Citation Articles by Goede, B. Articles by Thomm, M. Social Bookmarking                      What's this?

Protein-Protein Interactions in the Archaeal Transcriptional Machinery

BINDING STUDIES OF ISOLATED RNA POLYMERASE SUBUNITS AND TRANSCRIPTION FACTORS^*

Bernd Goede, Souad Naji, Oliver von Kampen, Karin Ilg, and Michael Thomm¹

From the Lehrstuhl für Allgemeine Mikrobiologie, Universität Kiel, am Botanischen Garten 1-9, 24107 Kiel and the Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universitaet Regensburg, 93053 Regensburg, Germany

Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.

Received for publication, May 31, 2006 , and in revised form, July 26, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental models.

¹ To whom correspondence should be addressed: Lehrstuhl für Mikrobiologie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg. Tel.: 0049-941-943-3160; Fax: 0049-941-943-2403; E-mail: michael.thomm{at}biologie.uni-regensburg.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Naji, M. G. Bertero, P. Spitalny, P. Cramer, and M. Thomm Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement Nucleic Acids Res., February 2, 2008; 36(2): 676 - 687. [Abstract] [Full Text] [PDF]

				S. Naji, S. Grunberg, and M. Thomm The RPB7 Orthologue E' Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation J. Biol. Chem., April 13, 2007; 282(15): 11047 - 11057. [Abstract] [Full Text] [PDF]