Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays.
Potyviruses express their genetic information from a genome length RNA as a single polyprotein, which is post-translationally processed by at least two different viral-encoded proteolytic activities. Since regulation of the expression of individual genes is not likely to occur at the transcriptional level, we sought to determine if post-translational regulation of gene expression was possible via differential proteolytic processing. Modulating the rate of cleavage at different gene product junctions could be a potential mechanism to regulate the kinetics of formation of specific gene products. We have examined the proteolytic processing of two tobacco etch virus (TEV) cleavage sites using a cell-free system in which synthetic polyprotein precursors were processed by the TEV 49-kDa proteinase. The amino acid sequences at these two sites contained both conserved and variable positions. The cleavage reaction at the TEV 50/71-kDa junction was characterized as "slow" (T1/2 = approximately 27 min) while the cleavage reaction at the 58/30-kDa junction was judged "fast" (T1/2 = approximately 5 min). Similar cleavage reactions were observed whether the cleavage site was in its natural polyprotein context or placed in a foreign position. The slow and fast cleavage sites could be interconverted by changing the nonconserved amino acid positions. The data suggest that the heptapeptide sequence proximal to a TEV cleavage site determines not only cleavage at a particular junction but influences the cleavage reaction profile in cell-free studies.
Dougherty
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WG
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Parks
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TD
D000595:Amino Acid Sequence; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D010942:Plant Viruses / Q000201:enzymology / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D012367:RNA, Viral; D014764:Viral Proteins; D010447:Peptide Hydrolases