NMR Studies on Termination/- Antitermination Complexes of HIV-1 Transcription
type: CreativeWork
description: After HIV enters the host cell the viral RNA is reverse transcribed into double stranded DNA and then integrated into the host genome. At this step, transcription of HIV RNA by Rpol II is tightly regulated by several cellular factors including NELF, DSIF, and pTEFb, and the virus encoded Tat protein. The work presented in this thesis is mainly focussed on understanding the mechanism of termination and antitermination of HIV-1 transcription. Some of the cysteines in the wild type HIV-1 Tat are required for the Zn2+ dependent interaction with pTEFb, but not needed for the binding of TAR RNA in vitro. These cysteines are readily oxidised and leading to the protein aggregation. Thus, a mutant, Tat-Cys-, lacking all cysteines was used for binding studies with TAR RNA. 1H-15N HSQC and 1H-1H TOCSY spectra were measured to monitor the interaction between Tat-Cys- and TAR RNA. The results presented indicate Tat-Cys- is binding to TAR RNA around the bulge region and is able to induce a conformational change in TAR RNA. This is further supported by observing a change in the CD signal at 265 nm upon addition of Tat-Cys- to TAR RNA. {1H}-15N steady state NOE experiments showed that the core region of Tat remains unstructured even in the complex with TAR RNA. Based on the NMR and CD spectroscopic data presented in this thesis, and data published by others it is tempting to speculate that the core and basic regions of Tat-Cys- remain unstructured upon binding to TAR RNA. Furthermore, the solution structure of the RNA binding domain of NELF-E, NELF-E RRM, was determined using multidimensional NMR spectroscopy. The data reveal that the structure of NELF-E RRM exhibits a babbab topology. The atomic coordinates were deposited in the protein data bank (pdb) under the accession code 2BZ2. RNA binding studies were performed with TAR RNA and various oligoribonucleotides. NMR studies with NELF-E RRM:TAR complexes indicate NELF-E RRM binds to various RNAs in a very similar manner but with d…
abstract: After HIV enters the host cell the viral RNA is reverse transcribed into double stranded DNA and then integrated into the host genome. At this step, transcription of HIV RNA by Rpol II is tightly regulated by several cellular factors including NELF, DSIF, and pTEFb, and the virus encoded Tat protein. The work presented in this thesis is mainly focussed on understanding the mechanism of termination and antitermination of HIV-1 transcription. Some of the cysteines in the wild type HIV-1 Tat are required for the Zn2+ dependent interaction with pTEFb, but not needed for the binding of TAR RNA in vitro. These cysteines are readily oxidised and leading to the protein aggregation. Thus, a mutant, Tat-Cys-, lacking all cysteines was used for binding studies with TAR RNA. 1H-15N HSQC and 1H-1H TOCSY spectra were measured to monitor the interaction between Tat-Cys- and TAR RNA. The results presented indicate Tat-Cys- is binding to TAR RNA around the bulge region and is able to induce a conformational change in TAR RNA. This is further supported by observing a change in the CD signal at 265 nm upon addition of Tat-Cys- to TAR RNA. {1H}-15N steady state NOE experiments showed that the core region of Tat remains unstructured even in the complex with TAR RNA. Based on the NMR and CD spectroscopic data presented in this thesis, and data published by others it is tempting to speculate that the core and basic regions of Tat-Cys- remain unstructured upon binding to TAR RNA. Furthermore, the solution structure of the RNA binding domain of NELF-E, NELF-E RRM, was determined using multidimensional NMR spectroscopy. The data reveal that the structure of NELF-E RRM exhibits a babbab topology. The atomic coordinates were deposited in the protein data bank (pdb) under the accession code 2BZ2. RNA binding studies were performed with TAR RNA and various oligoribonucleotides. NMR studies with NELF-E RRM:TAR complexes indicate NELF-E RRM binds to various RNAs in a very similar manner but with d…
author: Jampani, Nageswara Rao
date published: 2006-11-10
date modified: 2026-07-27T18:49:26Z
provider: Universität Bayreuth
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Universität Bayreuth