Biochem. J. (2006) 400
(449–456) (Printed in Great Britain)
Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription
Jampani N. RAO1, Liane NEUMANN1, Sabine WENZEL, Kristian SCHWEIMER2, Paul RÖSCH and Birgitta M. WÖHRL2
Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstrasse 30, D-95447 Bayreuth, Germany
The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has a-helical and b-strand elements adopting a babbab fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with Kd values in the low-micromolar range.
Key words: HIV termination/anti-termination, negative elongation factor E (NELF E), NMR structure, RNA-binding protein, transactivation-response element (TAR).
Abbreviations used: C12E5, penta(ethylene glycol)monododecyl ether; DSIF, DRB (5,6-dichloro-1b-D-ribofuranosylbenzimidazole)-sensitivity-inducing factor; DTT, dithiothreitol; FAM, 6-carboxyfluorescein; HIV-1, HIV type 1; HSQC, heteronuclear single-quantum coherence; IPTG, isopropyl b-D-thiogalactoside; MEXICO, measurement of exchange rates in isotopically labelled compounds; NELF, negative elongation factor; NOE, nuclear Overhauser effect; Pol II, polymerase II; pTEFb, positive transcription elongation factor b; RDC, residual dipolar coupling; RMSD, root mean square deviation; RRM, RNA-recognition motif; TAR, transactivation-response element.
1These authors contributed equally to this work.
2Correspondence may be addressed to either of these authors (email kristian.schweimer@uni-bayreuth.de or birgitta.woehrl@uni-bayreuth.de).
Structural co-ordinates for NELF (negative elongation factor) E RNA-recognition motif have been deposited in the Protein Data Bank under accession code 2BZ2.
The nucleotide sequence data for NELF (negative elongation factor) E RNA-recognition motif have been deposited in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number DQ885937.
Received 20 March 2006/9 August 2006; accepted 9 August 2006
Published as BJ Immediate Publication 9 August 2006, doi:10.1042/BJ20060421
The Biochemical Society, London ©2006
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