Figure 6.

Effect of rne hp2 on the physical lifetime of rne–lacZ reporter transcripts. Plasmids pEZΔ114–337T and pEZΔ114–337var2T were each introduced into an isogenic pair of lacZ⁻ E. coli host strains, CH1827 (rne⁺) containing the multicopy rne⁺ plasmid pRNE101, and CH1828 (ams-1). Cultures of each resulting strain were grown exponentially for several generations in LB medium at 37°C. At time intervals after transcription inhibition with rifampicin (200 μg/ml), total cellular RNA was isolated. Equal amounts of each RNA extract (4 or 8 μg) were then analyzed by primer extension using reverse transcriptase and a 5′ end-labeled DNA primer (5′-CCCAGTCACGACGTTGTAAAACG-3′) complementary to the lacZ coding region. The extension products were resolved by electrophoresis on a 4% polyacrylamide-urea gel beside a set of molecular size standards 900, 800, 700, and 600 nucleotides in length (M). The band corresponding to the rne–lacZ reporter transcripts is indicated. An additional band (✻) corresponding to a reporter RNA 48 nucleotides shorter at the 5′ end represents an apparent mRNA decay intermediate cleaved at a nonessential site within ss1 (Jain and Belasco 1995). The calculated size of the full-length primer extension products was 0.74 kb. The numbers above the autoradiogram lanes indicate minutes after transcription inhibition. Beneath each autoradiogram is a semilogarithmic plot of mRNA concentration as a function of time after rifampicin addition.