FIG. 3.

Transcriptional analyses of the metY-nusA-infB operon in different mutants. (A) Quantitation of metY-nusA-infB operon mRNAs in wild-type (wt) and nusA mutant strains by Northern blot analysis. Five micrograms of total RNA was subjected to electrophoresis in an agarose gel containing formaldehyde, transferred to a Hybond N filter, and probed with a radiolabeled PCR fragment corresponding to the p15a gene. The strains used (with the relevant genetic markers in parentheses) are indicated above the respective lanes. The sizes of the ³²P-end-labeled fragments of the 1-kb DNA ladder (GIBCO BRL Life Technologies Inc., Gaithersburg, Md.) are indicated. The 6.7-kb transcript results from read-through of the metYt₁ and metYt₂ terminators between metY and p15a and the infBt₃ terminator just upstream from rbfA, while the 4.8-kb transcript terminates at the infBt₃ terminator (Fig. 1). The amounts of these transcripts (determined by quantitation of the radioactivity using a PhosphorImager from Molecular Dynamics, Inc.) in the different nusA mutants were normalized to those for the nusA⁺ strain MW100. The read-through (RT) of the infBt₃ terminator was calculated as the amount of radioactivity in the 6.7-kb band divided by the sum of the radioactivity in the 4.8- and 6.7-kb bands. (B) Northern blot analysis showing the effect of deletions in the infBt₃ transcriptional terminator on the amount of the 6.7-kb transcript relative to that of the 4.8-kb transcript. (C) Identification of the 5′ end of the mRNA resulting from transcription initiation at a new promoter created by the insertion of IS2 in infB. Primer extension analyses of mRNA and DNA sequencing of a PCR fragment covering the 3′ part of infB and the 5′ part of rbfA from a wild-type strain were performed using a ³²P-end-labeled primer binding to positions −54 to −73 relative to the start codon of rbfA. The primer extension product obtained for strain JML125 (infB::IS2) corresponds to an mRNA 5′ end at the A 6 nt downstream from the −10 region of the proposed promoter.