Figure 4
Effect of the polypurine sequence on RNA stability. Panel A. pMYS694 carries the M. smegmatis -195/+27 DNA region (where +1 is the transcription initiation site from psigA [29]), containing the sigA promoter, upstream of lacZ in pSM128. The lacZ gene is preceded by its ribosome binding site (44 nt from the cloning site). pMYS690 carries the 437–531 region of M. smegmatis downstream of psigA, containing the wild type PPS (GAAAGGAAA); pMYS692 carries the same region with the indicated mutations in the PPS (CTCTGGAGG). pMYT733 and pMYT735 carry the 405–531 region of M. tuberculosis downstream of psigA. In pMYT733 carries the wild type PPS (GGAAGGAA), whereas in pMYT735 the PPS was substituted by CCTCCCTC. Beta-galactosidase activity and the half life of the lacZ mRNA were determined as indicated in the Methods section. Average beta-galactosidase activity in Miller Units of three to eight different replicas ± standard deviation is reported. The average relative increment in beta-galactosidase activity and in lacZ mRNA half life are also indicated. N.T. = not tested. Panel B. RNA was extracted from M. smegmatis strains mc2155(pMYS690) and mc2155(pMYS692), and primer extension performed with oligonucleotide 809, internal to lacZ, as described in Methods. The DNA sequence obtained with the same oligonucleotide on plasmid pMYS690 was run in the same gel.