A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV ta
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RESEARCH
Open Access
A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat Nina Verstraete1,2,3, Alona Kuzmina4, Gaelle Diribarne1,2,3, Van Trung Nguyen1,2,3, Lydia Kobbi1,2,3, Monika Ludanyi1,2,3,5, Ran Taube4 and Olivier Bensaude1,2,3,6*
Abstract Background: The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results: Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions: Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. Keywords: CDK inhibition, Genetic mapping of protein-protein interfaces, P-TEFb, Cyclin T, Hexim1, 7SK RNA
Background Transcription of most class II genes stops shortly after initiation because RNA polymerase II (RNAPII) is arrested by negative elongation factors (NELF, DSIF). Phosphorylation by the positive transcription elongation factor (P-TEFb) is required to counteract their effect and resume a productive transcription elongation [1]. In particular, recruitment of P-TEFb to the early transcription elongation complex is a critical step for productive HIV genome expression [2]. This recruitment relies on P-TEFb association with the Tat viral protein bound to the TAR domain of the nascent transcript and results in phosphorylation of the Negative Elongation Factors, NELF and DSIF, that prevent elongation of transcription. P-TEFb next phosphorylates the RNAPII Carboxy-Terminal Domain * Correspondence: [email protected] 1 Institut de Biologie de l’Ecole Normale Supérieure, Paris F-75005, France 2 UMR 8197, Centre National de la Recherche Scientifique, Paris F-75005, France Full list of author information is available at the end of the article
(CTD) and might thus contribute to recruit splicing, cleavage and polyadenylation factors for co-transcriptional pre-mRNA processin
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