TmRNA is required for correct timing of DNA replication in Caulobacter crescentus

Abstract

SsrA, or tmRNA, is a small RNA that interacts with selected translating ribosomes to target the nascent polypeptides for degradation. Here we report that SsrA activity is required for normal timing of the G(1)-to-S transition in Caulobacter crescentus. A deletion of the ssrA gene, or of the gene encoding SmpB, a protein required for SsrA activity, results in a specific delay in the cell cycle during the G(1)-to-S transition. The ssrA deletion phenotype is not due to accumulation of stalled ribosomes, because the deletion is not complemented by a mutated version of SsrA that releases ribosomes but does not target proteins for degradation. Degradation of the CtrA response regulator normally coincides with initiation of DNA replication, but in strains lacking SsrA activity there is a 40-min delay between the degradation of CtrA and replication initiation. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication.

FIG. 1.

SsrA peptide-tagging activity, processing, and the Caulobacter cell cycle. (A) Model for SsrA activity (22). A ribosome translating an mRNA becomes a substrate for SsrA RNA with the nascent polypeptide and tRNA still engaged. SsrA RNA charged with alanine on its tRNA-like 3′ end enters the ribosomal A site. Transpeptidation occurs to the alanine on SsrA, the mRNA is removed from the ribosome, and the translational reading frame switches to the mRNA-like portion of SsrA RNA. Translation of the SsrA reading frame adds a peptide to the incomplete protein and targets the protein for rapid proteolysis by several intracellular proteases. (B) Model for pre-SsrA transcription and processing into matureSsrA (21). A single transcript made from the ssrA gene folds into a structure similar to those of one-piece SsrA's, except that the 5′ and 3′ ends are in different parts of the molecule. Processing by nucleases produces the tRNA-like 5′ and 3′ ends, resulting in mature SsrA composed of a coding RNA and an acceptor RNA. (C) Comparison of the timing of major cell cycle events in wild-type Caulobacter and the ΔssrA strain. Cartoon diagrams of the Caulobacter cell cycle show the swarmer cell with a polar flagellum (curved line) and a single, nonreplicating chromosome (open circle), the stalked cell with a replicating chromosome (theta structure), and the predivisional cell with two completely replicated chromosomes (open circles).

FIG. 2.

Levels of SsrA RNA in wild-type (wt) Caulobacter and mutant strains, detected by Northern blotting. Equal quantities of total RNA were loaded in each lane and probed for SsrA RNA. Bands corresponding to pre-SsrA RNA, the coding RNA, and the acceptor RNA are indicated.

FIG. 3.

Replication initiation is delayed in SsrA-deficient strains. (A) Sample histogram showing the numbers of cells that had initiated replication for the wild-type (wt) and the ΔssrA strain at 45 min after synchronization. The regions of the histogram corresponding to one chromosome (1 X) and two chromosomes (2 X) are indicated at the top. (B) Histograms such as the one in panel A were used to determine the fraction of cells that had initiated replication at each time point in synchronized cultures of wild-type, ΔssrA, and ΔsmpB strains.

FIG. 4.

Expression of cell cycle-regulated proteins in mutant strains. Western blots of lysates from synchronized cultures of the wild type (wt) and ΔssrA strains were probed with antibodies for CtrA (A) and McpA (D). Blots such as these for wild-type, ΔssrA, and ΔsmpB strains (B and E) and plasmid-bearing strains (C and F) were quantified, normalized to the initial time point, and plotted to determine how levels change with respect to the cell cycle. Each curve represents average results of at least three synchronization experiments, and each error bar represents 1 standard deviation.