The dnaY gene of Escherichia coli encodes a transfer RNA, tRNA ARG/UCU. This is a most unusual tRNA gene for several reasons. (i) The primary transcript is a 180-190 nucleotide RNA which is processed to form the 77-nucleotide mature form. This mature form, however, can e folded into two alternative conformers, tRNA cloverleaf and a second, theoretically more stable conformer (Fig. 1). (ii) tRNA ARG/UCU is one of the rarest of tRNAs. Its cognate codons, AGA and AGG, are also extremely rare, there being only 94 among the 62,000 known E. coli codons. (iii) The presence of a typical regulatory protein binding site in the dnaY promoter region and the low level of expression of the gene suggest that dnaY is subject to repression. (iv) The dnaY gene is located adjacent to a gene, designated as dsy, identified at this time only as an open reading frame which encodes a 45,095 dalton protein detected in minicells. Based on its usage of rare codons, the Dsy protein is rare - perhaps a regulatory or replication protein. (v) The dsy gene contains 13 AGA/AGG codons. This extremely high number is about 15% of all the known AGA/AGGs. (vi) dnaY and dsy are transcribed in a convergent manner and the 3' ends of the transcripts overlap by at least 75 nucleotides. (vii) dnay is involved in replication. The evidence is convincing that a single based change in the mature RNA causes the replication defect, but it is not known if tRNA ARG/UCU or the alternative conformer is required or if the involvement is direct or indirect. Study of these two genes should reveal the function(s) of each and might described a previously unknown gene regulatory mechanism. First, it will be determined if a dnaY product participates directly or indirectly in replication by a mutational analysis to separate the translation and replication functions. An attempt will be made to develop an in vitro DNA synthesis assay dependent on dnaY RNA. Second, the mechanism of dnaY gene expression regulation will be determined, includig isolation of the dnaY repressor. Third, the function of the dsy gene will be determined by introducing conditional dsy mutant alleles into the chromosome. Fourth, the nature of the relationship between dnaY and dsy will be determied by measuring the overlap of their transcripts and testig the possibility that they regulate the expression of the other. Fifth, the possibility that two (or more) dnay conformers exist will be investigated and the secondary structure of each determined.
