Summary Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modeling, gel mobility shift, and footprint analyses identified two CsrA binding sites extending from positions 1–12 (BS1) and 44–55 (BS2) of the 198-nt flhDC leader transcript. flhD'-'lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5' end-dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD'-'lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5' end-dependent RNase E cleavage pathway.

Summary CsrA of Escherichia coli is an RNA binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine-Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ70-dependent (P2 and P5) and two σS-dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation.

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The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 59 ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.