RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.     

Alterations in tRNAs containing 2-methylthio-N6-(delta2-isopentenyl)-adenosine during growth of enteropathogenic Escherichia coli in the presence of iron-binding proteins.

Escherichia coli grown in chemically produced iron-deficient media have well characterized alterations in the chromatographic properties of tRNAs containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl) adenosine. The present report shows that similar tRNA alterations occur in enteropathogenic E. coli inhibited by human milk and bovine colostrum, the inhibited bacteria containing 10% or less of the normal tRNA species. Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in milk and colostrum reversed these changes which are probably due to a failure to methylthiolate the isopentenyladenosine. Although adding iron led to a rapid replacement of abnormal tRNA by the chromatographically normal species, and to a resumption of multiplication, the tRNA alterations are not directly related to the inhibition of growth. Strains of E. coli which grew normally in milk, colostrum and in defined media containing the iron-binding protein transferrin or ovotransferrin also contained about 90% of the abnormal species. Rapid conversion of abnormal tRNA to normal tRNA occurred on adding iron and in the absence of RNA synthesis. The tRNA changes are discussed in relation to their possible connection with both the adaptation of E. coli to growth under the iron-restricted conditions imposed by iron-binding proteins in tissue fluids and with bacterial pathogenicity.     

Crystallization and preliminary x-ray analysis of Escherichia coli peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase from Escherichia coli, a monomer of 21 kDa, was overexpressed from its cloned gene pth and crystallized by using polyethylene glycol as precipitant. The crystals are orthorhombic and have unit cell parameters a = 47.24 Å, b = 63.59 Å, and c = 62.57 Å. They belong to space group P2₁2₁2₁ and diffract to better than 1.2 Å resolution. The structure is being solved by multiple isomorphous replacement. © 1997 Wiley-Liss Inc.     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth^ts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth^ts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth^ts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.     

Protein release in recombinant Escherichia coli using bacteriocin release protein.

The effect of induction level of the bacteriocin release protein (BRP) on cell growth characteristics, protein expression, and protein release in a recombinant strain of Escherichia coli RR1 was investigated. Mitomycin C, the inducing agent, when added to the growth medium in moderate amounts (up to 200 ng/mL) was observed to enhance the release of periplasmic proteins from the cell to the fermentation broth substantially. The percentages of release of the proteins alpha-amylase and beta-lactamase were increased by factors of about 7 and 3, respectively, upon induction of the BRP. The percentage of alpha-amylase released into the broth increased from only about 5% to almost 50% with the aid of BRP. The cell growth curve and low extracellular activity of the cytoplasmic protein beta-galactosidase were indicative that cell lysis did not occur in an appreciable amount at a low induction level, with a mitomycin C concentration of less than 300 ng/mL.     

Effect of glutathione deficiency on the pool of CoA-glutathione mixed disulfide in Escherichia coli

The formic acid extracts of several glutathione-deficient strains of Escherichia coli have been assayed for the presence of the mixed disulfide of CoA and glutathione, CoASSG. Strains deficient in gamma-glutamyl-cysteine synthase (EC 6.3.2.2) produced only CoA dimer. Strains deficient in glutathione synthase (EC 6.3.2.3) produced the mixed disulfide of CoA and the gamma-glutamylcysteine dipeptide. The pool size of total CoA in the cell did not change significantly even in the absence of glutathione.     

Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase

The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome. In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification. The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA. Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity. Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20. These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.     

Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl-tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl-tRNA hydrolase could be solved at 1.2 A resolution. It indicates a single alpha/beta globular domain built around a twisted mixed beta-sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica. This similarity allowed the characterization by site-directed mutagenesis of several residues of the active site of peptidyl-tRNA hydrolase. These residues, strictly conserved among the known peptidyl-tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C-end of a neighbouring peptidyl-tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl-tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl-tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.     

Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli.

the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.     

Metabolism of 6-aminonicotinic acid in Escherichia coli.

A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-14C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6-aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.