hisT is part of a multigene operon in Escherichia coli K-12

C C Marvel, P J Arps, B C Rubin, H O Kammen, E E Penhoet, M E Winkler
1985 Journal of Bacteriology  
The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction
more » ... I-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated i300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid 4300 contained the hisT structural gene. Enzyme assays showed that plasmid q300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid *300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIlI site toward the ClaI site. Other subclones and derivatives of plasmid i300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIll-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu dl(lac Km) phage into plasmid i300 confirmed that the hisT gene is the downstream gene in the operon.
doi:10.1128/jb.161.1.60-71.1985 fatcat:phfeal4axrfqjimho2xvxfxgoe