Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the −10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the −10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⋅T base pair at the −11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a “master” role of the adenine base at −11 of the template strand in overall base unpairing. 2-AP at −11 did not inhibit the formation of RNA polymerase⋅promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, −11.

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation–transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3′ ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3′ RNA ends; thus the in vivo 3′ ends do not define termination sites. We predict generation of 3′ ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes. Significance Transcription termination by RNA polymerase in prokaryotes is well understood in contrast to similar mechanisms in higher organisms. Despite the in vitro occurrence of two types of demonstrable transcription termination events in prokaryotes at the end of transcription units, they are obscured in vivo in two ways: suppression of termination by traversing of the RNA polymerase through the termination sites when coupled to translation, or by further processing of the actual terminated RNA 3′ ends by RNases, as in eukaryotes.

The intracellular K⁺ level in bacteria is strictly controlled by K⁺ uptake and efflux systems. Among these, KdpFABC is a high-affinity K⁺ transporter system that is generally activated by the KdpDE two-component system in response to K⁺ limitation stress. However, the regulatory mechanism remains obscure in bacteria lacking the kdpDE genes. Here we report that the transcription of a kdpFABC operon is distinctively regulated by a cyclic diadenylate monophosphate (c-di-AMP) riboswitch located at the 5′-untranslated region of kdp transcript, and binding of c-di-AMP to the riboswitch promotes its intrinsic termination that blocks the kdpFABC transcription. Further, the intracellular c-di-AMP concentration was found to decrease under the K⁺ limitation stress, leading to transcriptional read-through over the terminator to allow kdpFABC expression. This regulatory element is found predominantly in the Bacillus cereus group and correlate well with the K⁺ and c-di-AMP homeostasis that affects a variety of crucial cellular functions.