DC Field | Value | Language |
---|---|---|
dc.contributor.author | Lee, Sang Soo | - |
dc.contributor.author | Kang, Changwon | - |
dc.date.accessioned | 2007-11-30T06:47:39Z | - |
dc.date.available | 2007-11-30T06:47:39Z | - |
dc.date.issued | 1993 | - |
dc.identifier.citation | J. Biol. Chem. 268, pp.19299-19304 | en |
dc.identifier.issn | 0021-9258 | - |
dc.identifier.uri | http://www.jbc.org/cgi/content/abstract/268/26/19299 | - |
dc.identifier.uri | http://hdl.handle.net/10203/2251 | - |
dc.description.abstract | Bacteriophage T7 and SP6 RNA polymerases and their promoters share a high degree of their primary structure homology, but each polymerase exclusively recognizes its own promoter sequence. To reveal the molecular basis of this specificity, 4 base pairs at positions -12, -10, -9, and -8 of the T7 promoter were substituted individually and multiply by SP6 promoter-specific base pairs, and 3 base pairs at -10, -9, and -8 of the SP6 promoter were replaced by T7 promoter-specific base pairs. Promoter activities of 28 sequences were measured in vitro with T7 and SP6 polymerases separately under optimal conditions at 6 mM MgCl2. Single and double substitutions at -12 and -10 do not significantly affect the T7 promoter activity, although they are almost exclusively conserved among T7 genomic promoters. Changes at -10 of SP6 promoter hardly affect the activity. However, any T7 variants that contain either or both changes at -9 and -8 show greatly reduced activity. Interestingly, the double substitution at -9 and -8 yields significant SP6 promoter activities and virtually no T7 promoter activity. Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 promoter activities, although they still show some SP6 promoter activities. However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity. No other SP6 variants show any T7 promoter activity. All these results indicate that the 2 base pairs at -9 and -8 of both the T7 and SP6 promoters are the primary (if not the only) determinants of specificity and that the hierarchy of importance of positions for promoter activity is -8, -9 > > -10 > -12. Also, a phylogenic relationship among the T3, T7, K11, and SP6 promoters is suggested based on dissimilarities in their sequences from -12 to -8. | en |
dc.language.iso | en_US | en |
dc.publisher | American Society for Biochemistry and Molecular Biology | en |
dc.subject | Bacteriophage T7 RNA polymerase | en |
dc.subject | Bacteriophage SP6 RNA polymerase | en |
dc.subject | Transcription initiation | en |
dc.subject | Promoter specificity | en |
dc.subject | RNA polymerase-Promoter interaction | en |
dc.subject | Nucleotide mutation | en |
dc.title | Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters | en |
dc.type | Article | en |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.