Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nifcluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nipK, nipT, nifY, nijE, nifV, nijX, nifU, niS, niJfV, nimW, nijZ, nfJM, and nipF. Although there are significant spatial differences, the identified A.vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A+T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.
Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW, nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nifW, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nifZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both nifZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.
The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of f luorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37°C on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of f luorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the Alu domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of f luorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.The signal recognition particle (SRP) is a ribonucleoprotein machine that delivers certain nascent polypeptides to specific recognition components on the cytoplasmic face of the endoplasmic reticulum membrane for translocation of secretory or membrane proteins (1, 2). The RNA component of the SRP contains two elements related to the human and rodent Alu families of interspersed repetitive DNA sequences connected by a unique sequence, the S domain (3-5). SRP RNA (Fig. 1) has an overall secondary structure that has been highly conserved during evolution of the Bacteria, Archaea, and Eukarya (6-9). SRP RNA associates with six proteins termed, in mammalian cells, SRP72, SRP68, SRP54, SRP19, SRP14, and SRP9 (10). The regions of SRP RNA bound by protein originally were identified by mild micrococcal nuclease digestion, which results in cleavage of the SRP into two subparticles containing the Alu and S domains, respectively (11). The Alu domain, which consists of approximately 100 nucleotides at the 5Ј end of SRP RNA base-paired with approximately 50 nucleotides at the 3Ј end, together with its associated proteins SRP9 and SRP14, comprises the translational arrest activity of SRP (12). The S domain consists of approximately 150 nucleotides of core SRP RNA sequence situated between the two Alu sequences and is bound by the remaining four proteins SRP19, SRP54, SRP68, and SRP72 (2). It is within the S do...
The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.
Abstract. The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These resuits demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA. cessing) was initially identified as an endoribonu-,Ik ~ clease that produces, in vitro, RNA primers for the initiation of mitochondrial DNA replication (Chang and Clayton, 1987a). When RNase MRP was subsequently discovered to be a ribonucleoprotein enzyme and its RNA (MRP RNA; see Fig. 1) component was sequenced (Chang and Clayton, 1987b), it turned out to be identical to a previously defined nucleolar RNA, termed 7-2 RNA (Reddy et al., 1981;Gold et al., 1989;Yuan et al., 1989). MRP RNA (7-2 RNA) is essential for RNase MRP enzymatic activity in vitro and is encoded by a nuclear gene (Chang and Clayton, 1987b;Yuan et al., 1989; Tooper and Clayton, 1990) that is transcribed by RNA polymerase III (Chang and Clayton, 1989). MRP RNA has been detected by in situ hybridization in both mitochondria and nucleoli of mouse cardiac myocytes (Li et al., 1994). The great majority of MRP RNA (7-2 RNA) in the cell fractionates with nucleoli (Reddy et al., 1981 Chu et al., 1994;Lygerou et al., 1994). In HeLa cells, nuclear MRP RNA is associated with large nucleolar structures sedimenting at N80 S (Kiss et al., 1992). MRP RNA-containing RNP particles can be immunoprecipitated by human To (Reddy et al., 1983), Th (Hashimoto and Steitz, 1983), and Wa (Reimer et al., 1988) sera (To, Th, and Wa representing patient codes for autoimmune sera that contain antibodies directed against the MRP RNA-containing RNP particle). The protein recognized by anti-To and anti-Wa antibodies is a ~40,000-mol wt nucleolar protein (Reimer et al., 1988). The 40,000-mol wt To autoantigen-binding domain was subsequently localized to nucleotides 21-64 of human MRP RNA (Yuan et al., 1991).In the present investigation, we have examined the subcellular localization of fluorescently tagged MRP RNA after microinjection into the nucleus of living cells. This method, which we term fluorescent RNA cytochemistry, is integrated with high re...
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