Steve Loechel

Nucleotide sequence of the MgPa (mgp) operon of Mycoplasma genitalium and comparison to the P1 (mpp) operon of Mycoplasma pneumoniae

The attachment of Mycoplasma genitalium and Mycoplasma pneumoniae to ciliated epithelium involves two surface proteins designated MgPa and P1, respectively. We have previously cloned and sequenced the P1 (mpp) operon of M. pneumoniae, and report here the use of P1-derived probes to clone and sequence a 10.4-kb region of M. genitalium DNA that, by analogy to the P1 operon, contains the MgPa (mgp) operon. The deduced amino acid sequences of the 29-kDa (ORF-1), MgPa (160-kDa) and 114-kDa (ORF-3) proteins of the MgPa operon show extensive homologies with those of the 28-kDa, P1 (170-kDa) and 130-kDa proteins, respectively, encoded by the P1 operon. The common features and homology of these operons are consistent with previous observations that the MgPa and P1 proteins share cross-reactive epitopes, as well as similar biological function. The gene order of the MgPa operon is ORF-1, MgPa, ORF-3, with intervening regions of 6 and 1 nt, respectively. A consensus ribosome-binding site (RBS) sequence is found before ORF-1 and a sequence indicative of a transcription terminator is located beyond ORF-3; the absence of such sequences adjacent to the MgPa gene suggests that the operon is transcribed as a polycistronic message. The RBS sequence is followed by sequences of dyad symmetry that have the potential to form two alternative stem-and-loop structures, which could be involved in controlling initiation of translation.

Analysis of the nucleotide sequence of the P1 operon of Mycoplasma pneumoniae

The attachment of virulent Mycoplasma pneumoniae to the ciliated epithelium of the respiratory tract involves a surface protein designated P1. Our previous determination of the nucleotide sequence of the P1 attachment-protein gene revealed that it is flanked by open reading frames (ORFs) and there is no obvious ribosome-binding site (RBS) or transcription termination sequence in the adjacent regions. We extended this analysis by cloning and sequencing the 18-kb region containing the P1 gene. This study indicates that the P1 gene is transcribed as part of a larger polycistronic message. The P1 operon is composed of the P1 gene and two predicted genes, designated ORF-4 and ORF-6. The gene order is ORF-4, P1, ORF-6 with intervening regions of 12 and 5 nt, respectively. ORF-4 and ORF-6 have respective coding capacities for proteins of Mr approximately equal to 28,000 and Mr approximately equal to 130,000. Putative promoter and RBS sequences which correspond closely to those found in Escherichia coli and Bacillus subtilis, as well as a sequence indicative of a transcription terminator, have been found in the flanking sequences. The transcription start point has been determined by primer extension of M. pneumoniae RNA.

Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcm1 and cyh2.

We placed a regulatory sequence derived from the GAL10 locus of Saccharomyces cerevisiae at various distances from the start sites of transcription of two yeast ribosomal protein genes, tcm1 and cyh2. The hybrid ribosomal protein genes were transcribed at wild-type levels in the presence of galactose. In the absence of galactose, the hybrid genes were transcribed either at a reduced level or essentially not at all. Yeast cells which transcribe the ribosomal protein genes at a reduced rate continued to grow, suggesting that enhanced translation of the ribosomal protein mRNA may permit an adequate rate of synthesis of the corresponding protein. Consistent with this suggestion is the finding that preexisting mRNA decayed at a reduced rate when transcription was halted abruptly by removal of galactose. Yeast cells unable to transcribe tcm1 or cyh2 without galactose did not grow. These conditional lethal strains demonstrate that the ribosomal proteins encoded by tcm1 and cyh2 are essential; furthermore, these strains are potentially useful for isolating mutations in the tcm1 and cyh2 proteins affecting their transport, assembly, or function.