Variants of lambda 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.

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.Proteolysis plays an important role in regulating the steadystate levels of many intracellular proteins. In addition, proteolysis is required for the degradation of misfolded proteins, the correct processing of secreted proteins, and survival under physiologically stressful conditions such as exposure to high temperature or nutrient deprivation. These varied proteolytic functions are carried out by many enzymes. In Escherichia coli, for example, over 20 different intracellular proteases have been identified (18).In this paper, we describe the identification and characterization of two E. coli genes, designated degQ and degS, which encode homologs of the DegP protease. DegP (HtrA) is a periplasmic serine protease that is required for E. coli growth at elevated temperatures (15,17,26) and is involved in the degradation of abnormal periplasmic proteins (25). The degQ gene is located immediately upstream of the degS gene, but each gene appears to be transcribed independently and neither is heat inducible. The degP gene, by contrast, is heat inducible (16). Using biochemical and genetic analyses, we show that the DegQ protein is a periplasmic serine endoprotease, which can functionally substitute for DegP under some conditions. The DegS protein cannot substitute for DegP but is required for normal bacterial growth. The degQ (hhoA) and degS (hhoB) genes have also recently been isolated as multicopy suppressors of the temperature/osmotic pressure-sensitive phenotype of an E. coli strain lacking another periplasmic protease (1). MATERIALS AND METHODSBacterial strains and plasmids. The E. coli strains used in this work are listed in Table 1. Bacteria were grown in LB medium (10 g of tryptone per liter, 5 g of yeast extract per liter, 5 g of NaCl per liter). Low-salt medium contains 5 g of tryptone per liter and 2.5 g of yeast extract per liter. High-salt medium is LB with an additional 15 g of NaCl per liter. Plates contained 1.5% agar. Ampicillin (100 g/ml), chloramphenicol (20 g/ml), and kanamycin (40 g/ml) were included as needed. Standard techniques were used for plasmid constructions.Subcloning and sequenci...

DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli.The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured. Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, ␤-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity. Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures. In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds. Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation. Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.Intracellular proteases play important regulatory roles and also serve essential housekeeping functions by removing damaged or misfolded proteins (18). Escherichia coli contains at least two cytoplasmic proteases, Lon and Clp, which function to degrade abnormal proteins (5). The biochemical properties of these ATP-dependent proteases have been studied extensively (4). Relatively little, however, is known about the biochemical or structural properties of proteases which may be responsible for degradation of misfolded or abnormal proteins in the periplasmic compartment of bacteria. Such a function has been attributed to the periplasmic protease DegP, which is also known as HtrA or protease Do (15,28,30,31). DegP is required for survival of E. coli at elevated temperatures, and mutations in the degP gene result in decreased degradation of chimeric membrane and periplasmic proteins (14,30,31). The temperature sensitivity of degP mutants is reduced in strains which release periplasmic proteins into the medium because of outer-membrane defects, as expected if DegP is required for the removal of misfolded proteins which may accumulate at high temperatures (16,30).Several groups recently identified another periplasmic protease of E. coli, DegQ (HhoA), which is homologous to DegP (2, 33). The DegQ and DegP proteins are of similar size (455 and 474 residues, respectively) and display approximately 60% sequence identity. Overproduction of DegQ suppresses the temperature-sensitive defect of a DegP Ϫ strain, suggesting that the two enzymes must be capable of degrading similar substrates (33). On the basis of sequence conservation, inhibition by diisopropyl fluorophosphate, and mutagenesis experiments, both enzymes seem to contain the Ser-His-Asp catalytic triad found in traditional serine proteases (29,...

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.

Advances in molecular genetics have identified several species of RNA that fail to translatehence the non-coding RNAs. The two major groups within this class of nucleic acids are microRNAs (miRNA) and long non-coding RNAs (lncRNA). There is growing body of evidence supporting the view that these molecules have regulatory effect on both DNA and RNA. The objective of this brief review is to explain the molecular genetic of these molecules, to summarise their potential as mediators of disease, and to highlight their value as diagnostic markers and as tools in disease management.