Eukaryotic transcription by RNA polymerase II is a highly regulated process and divided into three major steps: initiation, elongation, and termination. Each step of transcription is controlled by a number of cellular factors. Positive transcription factor b, P-TEFb, is composed of cyclin-dependent kinase 9 and a regulatory cyclin (T1/T2). P-TEFb promotes transcriptional elongation of RNA polymerase II by using the catalytic function of CDK9 to phosphorylate various substrates during transcription. P-TEFb is inactivated by sequestration in a complex with the Hexim1 protein and 7SK RNA. The structure of this inactive P-TEFb complex and the mechanisms controlling its equilibrium with the active complex are poorly understood. Here, we used a photoactive nucleotide, 4-thioU, to study the interactions between 7SK RNA and Hexim1. We identified a specific cross-link between nucleotide U30 of 7SK RNA and amino acids 210-220 of Hexim1, in the context of both a minimal RNA-binding site and a fully reconstituted 7SK/Hexim1/P-TEFb ribonucleoprotein complex. We show also that a minimal 7SK RNA hairpin comprising nucleotides 24-87 can bind specifically to Hexim1 in vivo. Our results demonstrate directly that the Hexim1 binding site is located in the 24-87 region of 7SK RNA and that the protein residues outside the basic domain of Hexim1 are involved in specific RNA interactions.

HIV-1 Tat protein regulates transcription elongation by binding to the 59 nt TAR RNA stem-loop structure transcribed from the HIV-1 5' long terminal repeat (5'-LTR). This established Tat-TAR interaction was used to investigate mRNA folding and RNA-protein interactions during early transcription elongation from the HIV-1 5'-LTR. Employing a new site-specific photo-cross-linking strategy to isolate transcription elongation complexes at early steps of elongation, we found that Tat interacts with HIV-1 transcripts before the formation of full-length TAR (TAR59). Analysis of RNA secondary structure by free energy profiling and ribonuclease digestion indicated that nascent transcripts folded into an alternative TAR RNA structure (TAR31), which requires only 31 nt to form and includes an analogous Tat-binding bulge structure. Functionally, TAR31, similar to TAR59, acts as a transcriptional terminator in vitro, and mRNA expression from TAR31-deficient HIV-1 5'-LTR mutant promoters is significantly decreased. Our results support a role for TAR31 in the control of HIV-1 mRNA transcription and we propose that this structure is important to stabilize the short early transcripts before the transcription complex commits for processive elongation. Overall, this study demonstrates that RNA folding during HIV-1 transcription is dynamic and that as the nascent RNA chain grows during transcription, it folds into a number of conformations that function to regulate gene expression. Finally, our results provide a new experimental strategy for studying mRNA conformation changes during transcription that can be applied to investigate the folding and function of nascent RNA structures transcribed from other promoters.