Post-transcriptional regulation of chloramphenicol acetyl transferase.

Abstract

The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) messenger ribonucleic acid (mRNA) encoded by plasmid pC194 was determined experimentally using gamma-32P-ATP-labeled run-off transcripts partially digested with T1 ribonuclease. By partial digestion of the in vitro transcripts with S1-, T1-, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions which contain the complementary sequences CCUCC, and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12 bp inverted complementary repeat sequence (-19.5 kcal/mol). The CAT mRNA was stable to digestion by T1 ribonuclease at the 4 guanosine residues in the Shine and Dalgarno sequence GGAGG, even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194, induced with chloramphenicol (CAM), contain about the same concentration of pulse labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the nonacetylatable CAM analog fluorothiamphenicol was tested using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro using Ba131 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by CAM required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis.