Analysis of 6S RNA-derived short pRNAs from Bacillus subtilis

6S RNA is a ubiquitous small non-coding RNA of ~200 nt that has been detected in all branches of the bacterial kingdom (Barrick et al, 2005). In E. coli, it interacts with the housekeeping RNA Polymerase (Es⁷⁰) in a growth phase-dependent manner, thereby inhibiting transcription from many s⁷⁰-dependent promoters during stationary phase (Wassarman et al, 2000). 6S RNA resembles an open promoter structure and is thus able to bind to the active site of Es⁷⁰. During exponential phase 6S RNA itself serves as a template for transcription of short oligonucleotides (so-called product RNAs = pRNAs) which results in the dissociation of the 6S RNA- Es⁷⁰ complex (Wassarman et al, 2006).

In contrast to most bacteria, Bacillus subtilis harbors two 6S RNA homologs termed 6S-1 and 6S-2. The main pRNA products transcribed from 6S-1 RNA products are 14 nt and 8/9 nt in length. Omission of ATP leads to production of 8/9-mers only, which in contrast to 14-nt pRNA transcripts do not promote 6S-1 RNA dissociation from RNA polymerase. Likewise, gel shift experiments have revealed that (i) the 8-nt pRNAs are not able to bind to 6S-1 RNA in a stable manner and that (ii) stable binding of the 14-nt pRNA to 6S-1 RNA prevents the RNA from binding to RNA polymerase. (iii) Intermediate-sized pRNA oligos (13-mer and 12-mer) have a substantially increased k_off relative to the 14-mer, but complexes with 6S-1 RNA are detectable and able to block binding to RNA polymerase.

Structure probing experiments have revealed that 6S-1 RNA : pRNA-14mer hybridization induces a structural rearrangement of the central bulge region of 6S-1 RNA, resulting in decreased affinity towards RNA Polymerase. Further experiments will address the question how the discontinuous length distribution of pRNAs is regulated and in turn contributes to the regulatory function of 6S RNA. Particularly a homonucleotide stretch found in many pRNAs from different bacteria has attracted our attention in this context.