DNA Sequences in gal Operon Override Transcription Elongation Blocks

Abstract The DNA loop that represses transcription from galactose ( gal ) promoters is infrequently formed in stationary-phase cells because the concentration of the loop architectural protein HU is significantly low at that state, resulting in expression of the operon in the absence of the gal inducer d -galactose. Unexpectedly, transcription from the gal promoters under these conditions overrides physical block because of the presence of the Gal repressor bound to an internal operator ( O I ) located downstream of the promoters. We have shown here that although a stretch of pyrimidine residues (UUCU) in the RNA:DNA hybrid located immediately upstream of O I weakens the RNA:DNA hybrid and favors RNA polymerase (RNAP) pausing and backtracking, a stretch of purines (GAGAG) in the RNA present immediately upstream of the pause sequence in the hybrid acts as an antipause element by stabilizing the RNA:DNA duplex and preventing backtracking. This facilitates forward translocation of RNAP, including overriding of the DNA-bound Gal repressor barrier at O I . When the GAGAG sequence is separated from the pyrimidine sequence by a 5-bp DNA insertion, RNAP backtracking is favored from a weak hybrid to a more stable hybrid. RNAP backtracking is sensitive to Gre factors, d -galactose, and antisense oligonucleotides. The ability of a native DNA sequence to override transcription elongation blocks in the gal operon uncovers a previously unknown way of regulating gal metabolism in Escherichia coli . It also explains the synthesis of gal enzymes in the absence of inducer for biosynthetic reactions.