Kenneth C. Keiler

Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.

Variants of λ repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.

Sequence Determinants of C-terminal Substrate Recognition by the Tsp Protease

Cytochrome b is not cleaved by the tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the C-terminal sequence WVAAA is added. Following randomization of the final three residue positions of this substrate, 54 different mutants with single residue substitutions were recovered. The steady-state expression levels of cytochrome variants bearing these mutant tails were similar in an E. coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp. Wild-type cytochrome b and seven variants, displaying a range of intracellular expression levels, were purified. These proteins were found to have the same T values in thermal denaturation experiments but to be cleaved by Tsp at rates differing by as much as 30-fold. Overall, the rates of Tsp cleavage of these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments. These results indicate that the C-terminal sequence of the cytochrome-b variants is important in determining their proteolytic fate in the cell and show that this degradation is mediated predominantly by Tsp. There are different selectivity rules at each of the three C-terminal positions. The identity of the C-terminal residue of the substrate, where small, uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most important in determining the rate of substrate cleavage by Tsp. Non-polar residues are also preferred at the second and third positions, but larger and more hydrophobic side chains are also acceptable at these positions in good substrates.

tmRNA Is Required for Correct Timing of DNA Replication in Caulobacter crescentus

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.

Identification of Active Site Residues of the Tsp Protease

In a search for active-site residues of the Tsp protease, 20 positions were individually mutated to alanine, the mutant strains were assayed for growth defects in vivo, and the purified proteins were assayed for proteolytic activity in vitro. Alanine substitutions at three positions, Ser-430, Asp-441, and Lys-455, result in inactive proteases that have structures and substrate-binding properties similar to wild type, suggesting that the side chains at these positions participate in catalysis. Replacing Ser-430 with cysteine results in a partially active protease, which is inhibited by cysteine-modifying reagents. Replacing Asp-441 with asparagine does not significantly affect activity. However, other residues, including histidine and arginine, cannot functionally replace Lys-455. These data are consistent with a serine-lysine dyad mechanism, similar to those proposed for the LexA-like proteases, the type I signal peptidases, and the class A β-lactamases.

Cell cycle‐regulated degradation of tmRNA is controlled by RNase R and SmpB

The  production  and  removal  of  regulatory  RNAs must be controlled to ensure proper physiological responses. SsrA RNA (tmRNA), a regulatory RNA conserved in all bacteria, is cell cycle regulated and is important for control of cell cycle progression in Caulobacter crescentus. We report that RNase R, a highly conserved 3′ to 5′ exoribonuclease, is required for the selective degradation of SsrA RNA in stalked cells. Purified RNase R degrades SsrA RNA in vitro, and is kinetically competent to account for all SsrA RNA turnover. SmpB, a tmRNA‐binding protein, protects SsrA RNA from RNase R degradation in vitro, and the levels of SmpB protein during the cell cycle correlate with SsrA RNA stability. These results suggest that SmpB binding controls the timing of SsrA RNA degradation by RNase R. We propose a model for the regulated degradation of SsrA RNA in which RNase R degrades SsrA RNA from a non‐tRNA‐like 3′ end, and SmpB specifically protects SsrA RNA from RNase R. This model explains the regulation of SsrA RNA in other bacteria, and suggests that a highly conserved regulatory mechanism controls SsrA activity.

Beyond ribosome rescue: tmRNA and co‐translational processes

tmRNA is a unique bi‐functional RNA that acts as both a tRNA and an mRNA to enter stalled ribosomes and direct the addition of a peptide tag to the C terminus of nascent polypeptides. Despite a reasonably clear understanding of tmRNA activity, the reason for its absolute conservation throughout the eubacteria is unknown. Although tmRNA plays many physiological roles in different bacterial systems, recent studies suggest a general role for trans‐translation in monitoring protein folding and perhaps other co‐translational processes. This review will focus on these new hypotheses and the data that support them.

Mechanisms of ribosome rescue in bacteria

Ribosomes that stall during translation need to be rescued to ensure that the protein synthesis capacity of the cell is maintained. Stalling arises when ribosomes become trapped at the 3′ end of an mRNA, which occurs when a codon is unavailable, as this leads to the arrest of elongation or termination. In addition, various factors can induce ribosome stalling in the middle of an mRNA, including the presence of specific amino acid sequence motifs in the nascent polypeptide. Almost all bacteria use a mechanism known as trans-translation to rescue stalled ribosomes, and some species also have other rescue mechanisms that are mediated either by the alternative ribosome-rescue factor A (ArfA) or ArfB. In this Review, I summarize the recent studies that have demonstrated the conditions that trigger ribosome stalling, the pathways that bacteria use to rescue stalled ribosomes and the physiological effects of these processes.

Subcellular localization of a bacterial regulatory RNA

Eukaryotes and bacteria regulate the activity of some proteins by localizing them to discrete subcellular structures, and eukaryotes localize some RNAs for the same purpose. To explore whether bacteria also spatially regulate RNAs, the localization of tmRNA was determined using fluorescence in situ hybridization. tmRNA is a small regulatory RNA that is ubiquitous in bacteria and that interacts with translating ribosomes in a reaction known as trans-translation. In Caulobacter crescentus, tmRNA was localized in a cell-cycle–dependent manner. In G1-phase cells, tmRNA was found in regularly spaced foci indicative of a helix-like structure. After initiation of DNA replication, most of the tmRNA was degraded, and the remaining molecules were spread throughout the cytoplasm. Immunofluorescence assays showed that SmpB, a protein that binds tightly to tmRNA, was colocalized with tmRNA in the helix-like pattern. RNase R, the nuclease that degrades tmRNA, was localized in a helix-like pattern that was separate from the SmpB-tmRNA complex. These results suggest a model in which tmRNA-SmpB is localized to sequester tmRNA from RNase R, and localization might also regulate tmRNA-SmpB interactions with ribosomes.

Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA‐tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.

tmRNA in Caulobacter crescentus Is Cell Cycle Regulated by Temporally Controlled Transcription and RNA Degradation

SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis. We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle. SsrA RNA abundance increases in late G(1) phase, peaks during the G(1)-S transition, and declines in early S phase, in keeping with the reported role for SsrA in the timing of DNA replication initiation. Cell cycle regulation of SsrA RNA is accomplished by a combination of temporally controlled transcription and regulated RNA degradation. Transcription from the ssrA promoter peaks late in G(1), just before the peak in SsrA RNA abundance. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. This degradation is surprising, since SsrA RNA is both highly structured and highly abundant. This is the first observation of a structural RNA that is cell cycle regulated.