Structural and Biochemical Studies of RNA Domains Involved in Regulation of Transcriptional Elongation of the HIV-1 retrovirus and Translational Recoding of the SARS Coronavirus

Abstract

Stable RNA structures interact with proteins at a frequency that allows for regulation of cellular and viral processes. Using a combination of Nuclear Magnetic Resonance (NMR), Small Angle X-Ray Scattering, Mass-Spectrometry and translational assays, I investigated two viral mRNA signals to understand what determines the frequency of recoding events that allows for fine-tuning of gene expression at the translational level. Here, I show that mutating loop residues that have the potential of forming long-range interactions with the major groove, and changing the pH of the translational mixture increases recoding efficiency of the SARS-Coronavirus pseudoknot (SARS-PK) in-vitro. In addition, I show that the SARS-PK is slightly flexible and the particular geometry of its junction induces a slight bent, which is sensitive to pH. Exploring the flexibility and ability of recoding signals to sample multiple conformations, I also present a minimal sequence required for optimal recoding efficiency in HIV-1, as well as, a set of mutations that partition the RNA into two conformations in-vitro and render the HIV-1 signal hyperactive in-vivo. Collectively, my results support a model where the SARS and HIV-1 recoding signals sample multiple conformations, but only one of them, the active signal, induces a fraction of the ribosomes to recode. In the second chapter, I investigated structural features of the 7SK- snRNA, which regulates the availability of the elongation factor b (p-TEFb) required for productive transcriptional elongation of HIV-1 and other cellular genes. I present a high-resolution structure of the first stem-loop of 7SK-snRNA (7SK-SL1) and show that one of its multiple pyrimidine bulges closely resembles the viral TAR-RNA. To understand how the RNA binding domain of the viral Tat protein binds 7SK-SL1 first and the viral TAR-RNA later, I also present a high-resolution structure of 7SK-SL1 in complex with the arginine-rich motif of Tat, and a binding comparison to the arginine-rich motifs of the cellular HEXIM and Brd4 by isothermal titration calorimetry (ITC) and NMR. Collectively, my results show structural mimicry between 7SK- snRNA and TAR-RNA, and that Tat’s arginine-rich motif displays structural advantages that may allow it to bind 7SK-SL1 first.