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The mRNA for CspA, a major cold shock protein in Escherichia coli , contains an unusually long (159 bases) 5′ untranslated region (5′-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5′-UTR of the cspA mRNA has a negative effect on its expression at 37°C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5′-UTR were constructed to examine the roles of specific regions within the 5′-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15°C. However, two mutations (Δ56-86 and Δ86-117) caused a substantial increase of β-galactosidase activity at 37°C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Δ2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of β-galactosidase. Interestingly, three mutations (Δ28-55, Δ86-117, and Δ118-143) caused poor cold shock induction of β-galactosidase. In particular, the Δ118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA , cspB , cspG , and cspI , and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.

In order to analyse the mechanism of cold shock induction of CspA, a major cold shock protein of Escherichia coli, deletion analysis of the cspA gene was carried out. It was found that (i) the AT-rich sequence (-47 to -38) upstream of the cspA -35 region may act as the UP element playing a crucial role in cspA transcription at both 37 degrees C and 15 degrees C; (ii) the unusually long 5'-UTR of the cspA mRNA has negative effects on cspA expression at 37 degrees C; and (iii) in contrast, the 5'-UTR exerts a positive effect on mRNA stabilization at low temperature. Furthermore, it was demonstrated that the 14 base downstream box (DB) locating 12 bases downstream of the initiation codon of the cspA mRNA and complementary to a region near the decoding region of 16S rRNA was essential for the mRNA translation during the growth lag acclimation phase immediately after cold shock. During this phase, translation of non-cold shock gene mRNAs is blocked, since they require cold shock-specific ribosomal factors for the formation of the translation initiation complex. It is proposed that DB in cold shock mRNAs allows the formation of a stable initiation complex at low temperature in the absence of the cold shock ribosomal factors.

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15 degrees C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15 degrees C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15 degrees C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.

Recently, asparaginyl endopeptidase was purified to homogeneity from jack bean (Canavalia ensiformis) seeds, and its NH2-terminal amino acid sequence was determined for 25 residues [Abe, Y. et al. (1993) J. Biol. Chem. 268, 3525-3529]. On the basis of this sequence information, we searched for seed cDNAs encoding this enzyme. Seven clones were obtained and sequenced. By combining four of them, we obtained a cDNA for a precursor of the enzyme containing the reported NH2-terminal sequence. The other three clones seemed to be for precursors of its isozymes. When the deduced amino acid sequences of these enzyme precursors were compared with those in the GeneBank, EMBL, and NBRF databases, only one protein was found with a homologous sequence. It was a precursor of hemoglobinase from a blood fluke (Schistosoma mansoni). Significant homology was observed only in the range of sequence for the mature form. Although hemoglobinase and asparaginyl endopeptidase behave as cysteine proteinases, their protein natures are distinct from either papain-type proteinases or clostripain. Various plant seeds have been reported to contain asparaginyl endopeptidases. This may be the first report, however, that deals with the primary structure of such a proteinase.

A cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3.5.1.14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney lambda gt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (M(r) 45,260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.

The nucleotide sequence of a cDNA coding for human aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1, EC 3.5.1.14) subunit has been determined. The amino acid sequence of human ACY-1 subunit deduced from its cDNA nucleotide sequence showed a high degree of identity (87.7%) with the corresponding protein from porcine [1], although the former is one amino acid residue longer than the latter. Of 12 histidine and 3 cysteine residues, conserved between the proteins from two species, some are anticipated to form the active site of ACY-1 as either catalytic residues or ligands for an essential Zn2+ atom.

Acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] is a tetrameric serine protease, which belongs to the oligopeptidase family and specifically removes acetyl amino acids from N-terminally acetylated peptides. By using diisopropyl fluorophosphate, we previously identified one of the residues comprising the catalytic triad of this enzyme as Ser587 [Miyagi, M. et al. (1995) J. Biochem. 118, 771-779]. To elucidate the other two residues forming the catalytic triad of this new serine-type protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with other oligopeptidases was substituted by site-directed mutagenesis, were expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme. All of the recombinant AAREs were estimated to have similar conformational and quaternary structures to the native porcine liver enzyme from their CD spectra and behavior on gel-filtration, but the mutants in which Ala587, Asn675, or Tyr707 was substituted for Ser587, Asp675, or His707, respectively, did not show detectable hydrolytic activity toward acetyl-L-methionyl L-alanine. These facts suggest that Ser587, Asp675, and His707 are essential residues for the AARE activity and comprise the catalytic triad of the enzyme in this order. Thus, AARE has been shown to have a protease-like domain in its C-terminal region, as do other proteins classified as members of the oligopeptidase family.

The nucleotide sequence of a cDNA coding for the human acylamino acid-releasing enzyme (AARE, also known as acylpeptide hydrolase) [EC 3.4.19.1] subunit has been determined. The amino acid sequence of human AARE subunit deduced from its cDNA nucleotide sequence showed a high degree of identity (91.5%) with both the corresponding proteins from the pig and the rat. The AARE cDNA shows 99.2% identity with a 3.3 kb cDNA transcribed from a locus (DNF15S2) on the short arm of human chromosome 3, whose deletion is associated with small cell lung cancer, taking into consideration that the sequence of the 3.3-kb cDNA previously reported was caused by misreading.

Enzymatical and chemical reactions that induce the peptide-bond cleavage at definite amino acid residues and so produce a limited number of fragments from proteins are essential tools in protein sequence analysis. Although several kinds of residue-specific proteases are now widely used for this purpose, the asparaginyl-bond specific protease has not been available yet. Carrington et al. (1985) and Bowles et al. (1986) have reported that concanavalin A, a lectin of jack bean (Canavalia ensiformis), is produced by post-translational proteolysis and transpeptidation (or ligation) at the carboxyl side of Asn-residues of its precursor. Their results suggest that the bean at least in its premature state contains an asparaginyl endopeptidase which may have a dual function, proteolysis and transpeptidation. We confirmed this suggestion by isolating it from commercially available jack-bean meal and showed its high utility in protein sequence analysis. Purification and characterization of this novel protease are described in the following.

Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] have been isolated from porcine liver cDNA lambda gt10 cDNA libraries and sequenced. Sequence analyses of the cDNA and several Achromobacter protease I-digested peptides of the purified protein revealed that porcine liver AARE consists of four identical subunits, and each comprising a single chain of 732 amino acids with acetylmethionine at the N-terminus.