The DNA sequence of a 2.3-kilobase segment of the E. coli hisT operon was determined. Analysis of the sequence indicated that the upstream gene in the operon encodes a 36,364-dalton polypeptide, which runs aberrantly on SDS-polyacryl ami de gel s. The distal hisT gene encodes the tRNA modification enzyme, pseudouridine synthase I, whichwias shown to have a polypeptide molecular mass of 30,399 daltons. The DNA sequence was consistent with the phenotypes and hisT expression of mutant operons. Analysis of the sequence and genetic complementation experiments demonstrated that the upstream and hisT genes are evolutionarily, structurally, and functionally unrelated; however, translation signals for the two genes overlap, which is consistent with genetic evidence suggesting translational coupling. Codon usage in the upstream gene is radically different from the hisT gene and may underlie the differential expression observed from the operon. Gene-inactivation experiments and Sl-mapping of in vivo transcripts indicated that the operon contains an additional upstream gene. S1-mapping experiments al so confirmed the presence of an internal promoter, which might be stringently controlled. Taken together, these results show that the structure of the hisT operon is complex and suggest that the operon might be regulated at sever levels.INTRODUCTI ON Maturation of stable RNA molecules occurs by multistep processing and modification of primary transcripts. A considerable number of biochemical and genetic studies have outlined the organization of genes that specify stable RNA molecules and the enzymatic cleavage steps involved in processing (reviewed in 1). In contrast, comparatively little is known about modification, even though this process is a dynamic, integral part of stable RNA biosynthesis in both prokaryotes and eukaryotes. In Escherichia coli, greater than 1% of the chromosome encodes the enzymes that modify tRNA and rRNA molecules (2). With the exception of the work of Bjork and his associates on tRNA-methyl transferase genes (sumimarized in 2), the structure and regulation of these genes are largely unknown. In addition, there is currently inadequate knowledge of the functions played by RNA modifications. Modifications undoubtedly play structural roles in stable RNA molecules, and they may influence the interac-© I R L Press Limited, Oxford, England.

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 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