Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription.
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 alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta 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 K(d) values in the low-micromolar range.
Rao
Jampani N
JN
Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstrasse 30, D-95447 Bayreuth, Germany.
Neumann
Liane
L
Wenzel
Sabine
S
Schweimer
Kristian
K
Rösch
Paul
P
Wöhrl
Birgitta M
BM
D020816:Amino Acid Motifs; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry* / Q000378:metabolism*
D009687:Nuclear Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; D012313:RNA