Recycling of a regulatory protein by degradation of the RNA to which it binds

Abstract

When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.

Fig. 1.

(A) Fold overexpression of trp operon genes from microarray experiments. The order of genes in the aromatic amino acid supraoperon is depicted. Promoters are indicated by hooked arrows. Numbers below genes indicate level of expression in the PNPase^- strain relative to wild type. Values are averages of four determinations, two measurements each from two independent RNA isolations. (B) Northern blot analysis of trp operon mRNA in wild-type and pnpA strains. Total RNA was separated on a formaldehydeagarose gel and probed with a uniformly labeled trpA antisense RNA. Migration of 23S and 16S ribosomal RNAs is indicated. Strains were grown in the presence (+) or absence (-) of tryptophan. (C) Northern blot analysis of trp operon mRNA in the pnpA mutant strain, carrying no plasmid (lane 1), vector plasmid + mtrB gene (lane 2), and vector plasmid alone (lane 3). The probe for both Northern blots shown was an antisense trpA probe.

Fig. 2.

Northern blot analysis of trp leader RNA. (A) Total RNA was isolated at various times after addition of rifampicin to inhibit new transcription. Above each lane is the time (minutes) after rifampicin addition. RNA was separated in a denaturing 6% polyacrylamide gel and probed with trp leader antisense RNA. Migration of full-length trp leader RNA and a protected fragment of trp leader RNA are indicated on the left. Sizes of 5′-end-labeled DNA fragments (in base pairs) are indicated to the right of the marker lane (M). (B) High-resolution separation of total RNA, followed by probing with trp leader antisense RNA. A DNA sequencing ladder was run in parallel as a size marker.

Fig. 3.

Analysis of 114-nt trp leader RNA in B. subtilis protein extracts. (A) Low-resolution polyacrylamide gels. Size marker DNA fragments were in lane M. Control (lane C) contained substrate RNA that was not incubated in the extract. Substrate RNA (migration indicated by arrows) was incubated in the extract without (lanes 1–3) or with (lanes 4–6) addition of 1.25 mM tryptophan. Additions were none (lanes 1 and 4), 1 mM Mn²⁺ (lanes 2 and 5), or 1 mM Mn²⁺ + 1 mM NaH₂PO₄ (lanes 3 and 6). Results using wild-type cell extract (Left) and pnpA mutant cell extract (Right) are shown. (B) High-resolution polyacrylamide gel. Samples loaded in lanes 1–6 are the same as those in lanes 1–6 in A Left. Migration of substrate RNA is indicated by the arrow at the left. A DNA sequencing ladder is at the right, with sizes (in nt) indicated.