An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of A repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.Within cells, different proteins are degraded with half-lives ranging from minutes to days (1). This heterogeneity implies that intracellular degradation is selective, but the mechanisms of this selectivity are incompletely understood. The C-terminal sequences of some proteins can have dramatic effects on their rates of degradation in Escherichia coli (2, 3). For example, the wild-type N-terminal domain of A repressor (residues 1-102) has the polar C-terminal amino acid sequence RSEYE102 and has a half-life in vivo of >10 hr. The rapidly degraded "#105" variant of this protein has the hydrophobic C-terminal sequence WVAAA102 and has a half-life of 15 min (3). To identify possible cellular component(s) responsible for the degradation of proteins with destabilizing C termini, we have purified an E. coli activity that specifically degrades the #105 variant but not the wild-type N-terminal domain. This activity resides in a single, purified polypeptide (Tsp, for tail-specific protease). We have cloned and sequenced the tsp gene.* Based on sequence comparisons, Tsp shows similarities to the human and bovine interphotoreceptor retinoid-binding proteins (IRBPs) but does not resemble any proteases in the protein sequence data base. MATERIALS AND METHODSAssays for Tsp Activity. Tsp activity in crude lysates and column fractions was assayed by measuring the degradation of 35S-labeled substrates (either the #105 variant or the wild-type N-terminal domain of A repressor) to products soluble in 10% (wt/vol) trichloroacetic acid (3). Reaction mixtures contained _9000 cpm of 35S-labeled substrate, a sample of the E. coli fraction in lysis buffer or column buffer, and 0.02% Nonidet P-40 in a volume of 50 p1. Reaction mixtures were incubated at 370C for 1 or 2 hr and were processed as described (3).In more purified fractions, degradation was assayed by the appearance of substrate digestion products following SDS/ PAGE. Wild-type or #105 protein (2 ,ug), which was purified by the method of Lim and Sauer (4), was mixed with a sample in a 10-1LI reaction volume. Reaction mixtures were incubated at 370C for 3-6 hr and were stopped by boiling for 3 min in Laemmli sample buffer (5). Samples were electrophoresed in SDS/16.5% polyacrylam...

Using the amino-terminal domain of k repressor as a model system, we show that residues in an unstructured region at the extreme carboxyl terminus of the protein are important for determining its proteolytic susceptibility in Escherichia coil Nonpolar amino acids are destabilizing when placed at the 5 carboxy-terminal residue positions, whereas charged and polar residues are stabilizing. The stabilizing effect of a single charged residue is greatest when it is at the terminal position and diminishes with increasing distance from the carboxyl terminus. The position of destabilizing sequences with respect to the free carboxyl terminus is important for their effect, but their distance from the folded portion of the protein is not important. Specific degradation of proteins with nonpolar carboxyl termini has been reconstituted in vitro using a partially pure, soluble fraction. This degradation is not ATP-dependent. Moreover, amino-terminal domain variants with nonpolar carboxyterminal residues are still rapidly degraded in strains that are deficient in proteolysis of abnormal proteins. These data suggest that the degradation of amino-terminal domain variants with nonpolar carboxy-terminal residues involves proteolytic components distinct from those known to be important for the turnover of unfolded proteins in E. coil

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free a-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.

The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini. To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain. Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of lambda repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro. Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E. coli.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids.The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new

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