Biomedical subjects

Yonglong Zhang

Publications and source records attributed to Yonglong Zhang.

9 recordsLinked to original sources

PubMed2006

Characterization of mRNA interferases from Mycobacterium tuberculosis.

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.

Amino Acid Sequence↗

PubMed2005

Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF.

MazF and MazE constitute a so-called addiction module that is critical for bacterial growth arrest and eventual cell death in response to stress. The MazF toxin was recently shown to possess mRNA interferase (MIase) activity, and acts as a protein synthesis inhibitor by cleaving cellular mRNA. As a cognate regulator, the short-lived antitoxin, MazE, inhibits MazF MIase activity and hence maintains the delicate homeostasis between these two components. In the present study, we have shown that the MazF homodimer contains two symmetric binding sites, each of which is capable of interacting with a MazE C-terminal peptide, MazEp(54-77). The slow exchange phenomenon between free and peptide-bound MazF on the NMR timescale indicates relatively high affinities for MazEp(54-77) at both sites (Kd,K'd < 10(-7) M). However, the observed sequential binding behavior suggests a negative cooperativity between the two sites (Kd < K'd). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd approximately 10(-5) -10(-6) M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.

Amino Acid Sequence↗

PubMed2005

Characterization of ChpBK, an mRNA interferase from Escherichia coli.

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues, and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5'-or3'-side of the A residue in ACY sequences (Y is U, A, or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2',3'-cyclic phosphate group at the 3'-end of the 5'-end product and a 5'-OH group at the 5'-end of the 3'-end product in a manner identical to that of MazF.

Amino Acid Sequence↗

PubMed2004

Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase.

MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5'-end of ACA sequences, yielding a 2',3'-cyclic phosphate at one side and a free 5'-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2'-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.

Base Sequence↗

PubMed2004

Interference of mRNA function by sequence-specific endoribonuclease PemK.

In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.

Base Sequence↗

PubMed2003

Characterization of the interactions within the mazEF addiction module of Escherichia coli.

In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin. The mazEF system is known as an addiction module located on the Escherichia coli chromosome. MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex. MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression. Here we show that the binding of purified (His)6MazE to the mazEF promoter DNA was enhanced by MazF. The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF-(His)6 complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain. The ratio of MazE to MazF(His)6 in the MazE-MazF(His)6 complex is about 1:2. Because both MazE and MazF-(His)6 exist as dimers by themselves, the MazE-MazF-(His)6 complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)6 dimers. The interaction between MazE and MazF was also characterized with the yeast two-hybrid system. It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF. Site-directed mutagenesis at this region revealed that Leu55 and Leu58 play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA. The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF.

Amino Acid Sequence↗

PubMed2003

Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities.

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.

Adenosine Triphosphate↗

PubMed2003

MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli.

Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.

Bacterial Proteins↗

PubMed2002

[Molecular characteristics of beta-lactamase from Klebsiella pneumoniae].

OBJECTIVE: To study the molecular characteristics of beta-lactamase from Klebsiella pneumoniae. METHODS: Beta-lactamase was prepared from strains of Klebsiella pneumoniae isolated clinically for which the minimal inhibitory concentration (MIC) of cefoperazone had been >or= 8 mg/L and then was reduced by 50% by the addition of sulbatam. The isoelectric points of different beta -lactamases were examined by LKB2117 Multiphor II Electrophoresis System. The molecular weights were examined by polyacrylamide gel electrophoresis. PCR was used to detect the TEM-type genes. The PCR products were sequenced by chemiluminescence. RESULTS: All four strains of Klebsiella pneumoniae produced more than two kinds of beta-lactamase. TEM-type genes were amplified from all strains. 97% - 98% of the DNA sequence and amino acid sequence of the TEM-type enzymes from 2 strains of Klebsiella pneumoniae were identical with those of TEM-1 enzyme. The isoelectric points of the beta-lactamases ranged from 5.4 to 9.30 and the molecular weights were between 23.0 and 43.0 KD. The beta-lactamases from Klebsiella pneumoniae 99592 and K. pneumoniae 99607 hydrolysed ceftazidine, cefotaxime, and ceftriaxone. The beta-lactamase from K. pneumoniae 99595 had stronger activity against ceftazidime, ceftaxime, and ceftriaxone than that from K. pneumoniae 99607. CONCLUSION: Klebsiella pneumoniae produces TEM-type extended spectrum beta -lactamase against ceftazidine, ceftaxime, and ceftriaxone.

Isoelectric Point↗