Ribosomal protein L20 controls expression of the Bacillus subtilis infC operon via a transcription attenuation mechanism

In contrast to Escherichia coli no molecular mechanism controlling the biosynthesis of ribosomal proteins has been elucidated in Gram-positive organisms. Here we show that the expression of the Bacillus subtilis infC-rpmI-rplT operon encoding translation factor IF3 and the ribosomal proteins L35 and L20 is autoregulated by a complex transcription attenuation mechanism. It implicates a 200-bp leader region upstream of infC which contains two conserved regulatory elements, one of which can act as a transcription terminator. Using in vitro and in vivo approaches we show that expression of the operon is regulated at the level of transcription elongation by a change in the structure of the leader mRNA which depends upon the presence of ribosomal protein L20. L20 binds to a phylogenetically conserved domain and provokes premature transcription termination at the leader terminator. Footprint and toeprint experiments support a regulatory model involving molecular mimicry between the L20-binding sites on 23S rRNA and the mRNA. Our data suggest that Nomura's model of ribosomal protein biosynthesis based on autogenous control and molecular mimicry is also valid in Gram-positive organisms

Régulation de l'expression de protéines ribosomiques chez Bacillus subtilis (cas de l'opéron infC)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Effect of L20 overproduction on the transcription profile of the operon

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> Northern analysis of total RNA of a wild-type strain harboring plasmid pHML17 carrying an IPTG inducible copy of the (L20) gene was performed using two different probes. () RNA was separated on a 0.8% agarose gel and probed with an specific probe including the entire leader sequence (see Materials and methodssection). () RNA separated on a 8% polyacrylamide gel was probed with oligonucleotide HP1080 complementary to positions +8 to +34 of the leader. Where indicated IPTG (1 mM) was added to mid-log cultures for 20 min prior to isolation of the RNA. RT = read-through transcript, T = terminated transcript. The positions of the molecular size markers are indicated. Percentages of read-through transcripts are indicated below the gel

Effect of leader mutations and the presence of L20 on the transcription profile

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> Single round transcription assays were performed on PCR templates comprising the promoter and 5′ noncoding region of using RNA polymerase. RT and T on the left side indicate read-through or prematurely terminated leader transcripts. The size of the marker fragments in bases is indicated. Numbers at the bottom indicate the percentage of read-through transcripts (%RT = (RT/(T + RT) × 100). () Templates were wild type (wt) or contained the A, B or A + B mutations depicted in . () Where indicated proteins were added in 50-fold molar excess over the template during the elongation phase of the single-round transcription assay. The L20 (Bs L20) and L17 (Bs L17) r-proteins were native, L20 (Ec L20) only contained the C-terminal half of the protein

RNase probing of the leader mRNA structure in the absence or presence of L20 protein

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> An leader transcript (nts 1–168) was subjected to cleavage by RNases V1 or T1. Where indicated purified L20 protein was added prior to RNase cleavage. () Cleavages by RNase V1 and RNase T1 are shown on the leader mRNA structure. Colors indicate the change in cleavage efficiency observed in the presence of L20: black (no change), red (increase), blue (decrease). The numbers next to the symbols locate the corresponding cleavages on the gels (boxes). Shaded and encircled nucleotides correspond to positions conserved at the L20-binding site on 23S rRNA as shown in . () Cleavages on an unlabeled transcript were detected by primer extension with labeled oligonucleotide HP697 (positions 168–141 on leader). () Cleavages on a 5′ labeled transcript were analyzed directly