Identifying molecular interactions important for PKR function
Studies involved with the over-expression of full length PKR an d1-170 PKR in-vivo, although successful, did not obtain sufficient quantities of protein for crystallography. Protein studies were continued with the use of in-vitro transcription/translation which showed no PKR inhibitor function in an...
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ndltd-bl.uk-oai-ethos.bl.uk-6962532018-04-04T03:30:56ZIdentifying molecular interactions important for PKR functionGale, Benjamin William1997Studies involved with the over-expression of full length PKR an d1-170 PKR in-vivo, although successful, did not obtain sufficient quantities of protein for crystallography. Protein studies were continued with the use of in-vitro transcription/translation which showed no PKR inhibitor function in any of the bluetongue viral proteins screened. As well as this, no definitive RNA- kinase interaction was found. Further studies examining the occurrence of a doublet within 1-170 PRK proved that the phenomenon was not the result of residue serine 6 acting as a phosphorylation site or leaky scanning.;RNA binding studies were carried out in order to characterise the dsRNA binding domain of PKR. These studies successfully mapped the region to between PKR residues 10 and 170. Further Poly(I).Poly(C) binding analysis showed the N-terminal boundary to be located to between residues 11 and 30, with the C-terminal boundary between residues 160 and 170. Site directed mutagenesis was undertaken to investigate the contribution of individual amino acids on dsRNA binding within this region. The highly conserved K69 and K159 residues were shown not to be critical for dsRNA binding although a contribution to a positively charged cluster is likely. Mutation of the absolutely conserved A67, A68, A157 and A158 residues did not prevent dsRNA binding or appear to alter the predicted structure of the domain as determined by modelling analysis. Consequently, these conserved residues have been predicted to play a role in maintaining the structure of the domain or more speculatively allowing PKR-PKR dimerisation.572.8University of Leicesterhttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696253http://hdl.handle.net/2381/29804Electronic Thesis or Dissertation |
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572.8 Gale, Benjamin William Identifying molecular interactions important for PKR function |
description |
Studies involved with the over-expression of full length PKR an d1-170 PKR in-vivo, although successful, did not obtain sufficient quantities of protein for crystallography. Protein studies were continued with the use of in-vitro transcription/translation which showed no PKR inhibitor function in any of the bluetongue viral proteins screened. As well as this, no definitive RNA- kinase interaction was found. Further studies examining the occurrence of a doublet within 1-170 PRK proved that the phenomenon was not the result of residue serine 6 acting as a phosphorylation site or leaky scanning.;RNA binding studies were carried out in order to characterise the dsRNA binding domain of PKR. These studies successfully mapped the region to between PKR residues 10 and 170. Further Poly(I).Poly(C) binding analysis showed the N-terminal boundary to be located to between residues 11 and 30, with the C-terminal boundary between residues 160 and 170. Site directed mutagenesis was undertaken to investigate the contribution of individual amino acids on dsRNA binding within this region. The highly conserved K69 and K159 residues were shown not to be critical for dsRNA binding although a contribution to a positively charged cluster is likely. Mutation of the absolutely conserved A67, A68, A157 and A158 residues did not prevent dsRNA binding or appear to alter the predicted structure of the domain as determined by modelling analysis. Consequently, these conserved residues have been predicted to play a role in maintaining the structure of the domain or more speculatively allowing PKR-PKR dimerisation. |
author |
Gale, Benjamin William |
author_facet |
Gale, Benjamin William |
author_sort |
Gale, Benjamin William |
title |
Identifying molecular interactions important for PKR function |
title_short |
Identifying molecular interactions important for PKR function |
title_full |
Identifying molecular interactions important for PKR function |
title_fullStr |
Identifying molecular interactions important for PKR function |
title_full_unstemmed |
Identifying molecular interactions important for PKR function |
title_sort |
identifying molecular interactions important for pkr function |
publisher |
University of Leicester |
publishDate |
1997 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696253 |
work_keys_str_mv |
AT galebenjaminwilliam identifyingmolecularinteractionsimportantforpkrfunction |
_version_ |
1718619785892200448 |