To screen, isolate, and characterize bacterial populations producing microcin J25, we report here two rapid, reliable, and sensitive methods, using polymerase chain reaction and colony blot hybridization with a digoxigenin-labelled probe. A sample of 26 Escherichia coli strains isolated from poultry intestinal contents was evaluated to detect the sequence of mcjA, the gene encoding the MccJ25 precursor. The two molecular techniques were compared with the commonly used cross-immunity tests. They generate accurate data with no obvious cross-reactions with other microcins. The results display that the producers of MccJ25 were widely distributed in the poultry intestinal habitat. The applications of these molecular methods will be useful in future studies of microcinogenic populations, and thus contribute to understand the relationships within the complex intestinal microbial ecosystem.
Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5'UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3'UTR. Despite the weak requirement for a long 5'UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation.
ABSTRACT Escherichia coli LR05, in addition to producing MccB17, J25, and D93, secretes microcin L, a newly discovered microcin that exhibits strong antibacterial activity against related Enterobacteriaceae , including Salmonella enterica serovars Typhimurium and Enteritidis. Microcin L was purified using a two-step procedure including solid-phase extraction and reverse-phase C 18 high-performance liquid chromatography. A 4,901-bp region of the DNA plasmid of E. coli LR05 was sequenced revealing that the microcin L cluster consists of four genes, mclC , mclI , mclA , and mclB . The structural gene mclC encoded a 105-amino-acid precursor with a 15-amino-acid N-terminal extension ending with a Gly-Ala motif upstream of the cleavage site. This motif is typical of the class II microcins and other gram-positive bacteriocins exported by ABC transporters. The mclI immunity gene was identified upstream of the mclC gene and encodes a 51-amino-acid protein with two potential transmembrane domains. Located on the reverse strand, two genes, mclA and mclB , encoded the proteins MclA and MclB, respectively. They bear strong relatedness with the ABC transporter proteins and accessory factors involved in the secretion of microcins H47, V, E492, and 24. The microcin L genetic system resembles the genetic organization of MccV. Furthermore the MccL primary structure has been determined. It is a 90-amino-acid peptide of 8,884 Da with two disulfide bridges. The N-terminal region has significant homologies with several gram-positive bacteriocins. The C-terminal 32-amino-acid sequence is 87.5% identical to that of MccV. Together, these results strongly indicate that microcin L is a gram-negative class II microcin.
The regulation of HIV expression is controlled by the activity of the Long Terminal Repeat (LTR). Trans-activation by the virally encoded Tat protein is one of the main mechanisms of LTR activation. Tat binds to its target, TAR RNA, and cellular proteins that bind the LTR, Tat, or TAR RNA are important components of the trans-activation process. We will review the factors that have been characterized for a possible involvement in this mechanism. Whereas LTR binding proteins consist of Sp1 and TBP, a large number of factors that bind TAR RNA have been isolated. We have previously cloned two of them by RNA probe recognition: TRBP and La. We have shown that the in vitro and in vivo binding of TRBP to TAR RNA correlates with a constant expression of the protein during HIV-1 infection. Several proteins that interact with Tat have mainly positive, but some negative, effects on trans-activation. Genetic studies have defined that human chromosome 12 encodes a protein that will allow trans-activation in rodent cells. The binding and the functional data about these proteins suggest sequential steps for the Tat trans-activation mechanism. Each of these intracellular molecular events could be the target for molecular intervention against the virus.
Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30–34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted α-helical structure. A deletion mutant lacking the 10 C-terminal aa (ΔC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30–34 kDa isoforms). In contrast, the ΔC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant ΔC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.
ABSTRACT The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH 2 -terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.
Double‐stranded RNA‐binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV‐1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24‐amino‐acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA‐binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 × 10 −8 m , whereas dsRBD1 binds RNA minimally. Binding analysis of 25‐amino‐acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC‐rich TAR‐mimic RNA. Whereas a 25‐mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15‐amino‐acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA‐binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR‐helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.
TRBP1 and TRBP2 are isoforms of a double-stranded RNA-binding protein that differ in their N-terminal end and were each identified by binding to human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive RNA. TRBP1 and TRBP2 also bind and modulate the function of the double-stranded RNA-activated protein kinase, protein kinase R. Both proteins increase long terminal repeat expression in human and murine cells, and their gene has been mapped to human chromosome 12. We have isolated and characterized the complete tarbp2 gene (5493 bp) coding for the two TRBP proteins. Two adjacent promoters initiate transcription of alternative first exons for TRBP1 and TRBP2 mRNAs that are spliced onto common downstream exons. TRBP2 transcription and translation start sites are localized within the first intron of TRBP1. TRBP promoters are TATA-less but have CCAAT boxes, a CpG island, and several potential binding sites for transcriptional factors. Promoter deletion analysis identified two regions from position −1397 to −330 for TRBP1 and from position −330 to +38 for TRBP2 that are important for promoter function. TRBP2 promoter activity was expressed at a higher level compared with TRBP1 promoter. In addition, a specific down-regulation of TRBP1 and TRBP2 promoter activity was identified in human astrocytic cell line U251MG compared with HeLa cells. This minimal TRBP promoter activity may account for minimal HIV-1 replication in astrocytes.
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