Nucleic Acids Research Advance Access originally published online on August 18, 2006
Nucleic Acids Research 2006 34(15):4278-4292; doi:10.1093/nar/gkl499
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Nucleic Acids Research, 2006, Vol. 34, No. 15 4278-4292
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Dynamics of nascent mRNA folding and RNA–protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School 364 Plantation Street, Worcester, MA 01605-2324, USA
*To whom correspondence should be addressed. Tel: +1 508 856 6216; Fax: +1 508 856 6696; Email: tariq.rana{at}umassmed.edu
Received February 24, 2006. Revised May 22, 2006. Accepted June 28, 2006.
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.
Present addresses: Sara N. Richter, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via Gabelli 63, 35121 Padova, Italy Ping Zheng, Roche Pharmaceuticals, Nutley, NJ, USA
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