NMR Studies on Termination/- Antitermination Complexes of HIV-1 Transcription
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 different affinities. Mapping of chemical shift perturbations on the structure of NELF-E RRM indicate that the RNA binding interface is mainly located on the b-sheet surface. Large chemical shift perturbations are observed for the residues located in the flexible C-terminal region of NELF-E RRM and in the loop between b3 and a2. Among the various RNAs tested, TAR49-57 displayed the highest affinity to NELF-E RRM. Further structural characterisation of the NELF-E RRM:TAR49-57 complex revealed that the C-terminal region of NEFL-E RRM adopts a small 310 helix around the b3-a2 loop and is stabilised by several hydrophic interactions. The C-terminal region of NELF-E RRM in the RNA bound conformation is very close to the RNA binding interface and could be involved in the RNA binding. However, further structural characterisation of NELF-E RRM in the complex with RNA will be needed to fully understand the mechanism of RNA recognition.
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