Efficient transcription of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase obtained by a new procedure [corrected].
The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion with ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66.
Deiman
B A
BA
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Séron
K
K
Jaspars
E M
EM
Pleij
C W
CW
D000914:Antibodies, Viral / Q000276:immunology; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D005990:Glycerol / Q000737:chemistry; D008836:Micrococcal Nuclease / Q000378:metabolism; D012343:RNA, Transfer; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D017806:Tymovirus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D000914:Antibodies, Viral; D014764:Viral Proteins; D012343:RNA, Transfer; D012324:RNA-Dependent RNA Polymerase; D008836:Micrococcal Nuclease; D005990:Glycerol