A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

Transcript decay mediated by RNase III in Borrelia burgdorferi.

The causative agent of Lyme disease, Borrelia burgdorferi, requires shifts in gene expression to undergo its natural enzootic cycle between tick and vertebrate hosts. mRNA decay mechanisms play significant roles in governing gene expression in other bacteria, but are not yet characterized in B. burgdorferi. RNase III is an important enzyme in processing ribosomal RNA, but it also plays a role in mRNA decay in many bacteria. We compared RNA decay profiles and steady-state abundances of transcripts in wild-type Borrelia burgdorferi strain B31 and in an RNase III null (rnc-) mutant. Transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), a flagellar protein (flaB), and a translational regulator (bpuR) decayed more rapidly in the wild-type strain than in the slow growing rnc- mutant indicating that RNA turnover is mediated by RNase III in the bacterium that causes Lyme disease. Additionally, in wild type bacteria, RNA decay rates of rpoS, rpoN, ospA, ospC, bpuR and dbpA transcripts are only modestly affected by changes in the osmolarity.

Measurements of mRNA degradation in Borrelia burgdorferi.

The importance of gene regulation in the enzootic cycle of Borrelia burgdorferi, the spirochete that causes Lyme disease, is well established. B. burgdorferi regulates gene expression in response to changes in environmental stimuli associated with changing hosts. In this study, we monitored mRNA decay in B. burgdorferi following transcriptional arrest with actinomycin D. The time-dependent decay of transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), outer surface lipoproteins (ospA and ospC), and a flagellar protein (flaB) have different profiles and indicate half-lives ranging from approximately 1 min to more than 45 min in cells cultured at 35°C. Our results provide a first step in characterizing mRNA decay in B. burgdorferi and in investigating its role in gene expression and regulation.

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA.

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.

Translational repression mechanisms in prokaryotes.

Translational repression results from a complex choreography of macromolecular interactions interfering with the formation of translational initiation complexes. The relationship between the rate and extent of formation of these interactions to form repressed mRNA complexes determines the extent of repression. A novel analysis of repression mechanisms is presented here and it indicates that the reversibility of repressed complex formation influences the steady state balance of the distribution of translationally active and inactive complexes and therefore has an impact on the efficiency of repression. Reviewed here is evidence for three distinct translational repression mechanisms, regulating expression of the transcription factor sigma32, threonine tRNA synthetase and ribosomal proteins on the alpha operon in Escherichia coli. Efficient regulation of expression in these systems makes use of specific mRNA structures in quite different ways.

Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit.

Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses. DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions. In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1). DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit. The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence. In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits. We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment. The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.