The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

Kempsell, Karen E.; Ji, Yuan-En; Estrada, Iris; Colston, M. Joseph; Cox, Robert A.

doi:10.1099/00221287-138-8-1717

Cited by 88 publications

(88 citation statements)

References 35 publications

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“…For expression analysis in E. coli, the M. tuberculosis murA gene was amplified from λEMBL3\TB3 (a gift from Dr J. Colston, NIMR, Mill Hill, London) (Kempsell et al, 1992) using primers MurA5h and MurA3h (Table 1) to generate clone MurA2T6. MurA5h is homologous to the 5h-terminus of the murA gene upstream sequence and also spans the initiation codon, substituting nucleotides k2 to j1 with CCA (depicted in bold italics in Table 1) to generate an ATG start codon and unique NcoI cutting site at the 5h-terminus of the coding region.…”

Section: Methodsmentioning

confidence: 99%

Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis The EMBL accession number for the sequence in this paper is X96711.

Smet¹,

Kempsell²,

Gallagher³

et al. 1999

Microbiology

131

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Mycobacterium tuberculosis has innate resistance to a range of broadspectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the M. tuberculosis MurA enzyme might confer resistance. In Escherichia coli, fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the M. tuberculosis MurA. In the present study, it is demonstrated that the wild-type M. tuberculosis MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an M. tuberculosis enzyme that is unstable during constitutive expression.

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Section: Methodsmentioning

confidence: 99%

Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis The EMBL accession number for the sequence in this paper is X96711.

Smet¹,

Kempsell²,

Gallagher³

et al. 1999

Microbiology

131

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“…The region spanning boxA and boxC shows 100% sequence conservation between rrn leader regions of different species of mycobacteria, suggesting an essential function, whereas the boxB element, comprising a stem-loop structure, has no strict sequence conservation between species or between rrn operons. The motifs are all found within the first half of M. tuberculosis rrn leader sequence (23). In this study we investigated interactions between recombinant M. tuberculosis NusA and the M. tuberculosis rrn leader region in vitro and identified a sequence-specific, high-affinity interaction between the rrn nut site and NusA.…”

mentioning

confidence: 99%

“…tuberculosis harbors a single, nonredundant rrn operon, the regulation of which therefore comprises an obvious target for new therapeutic agents. The M. tuberculosis rrn leader and spacer regions contain sequences with homology to the E. coli rrn antitermination motifs, boxB, boxA, and boxC, with similar characteristics and organization (11,23). The region spanning boxA and boxC shows 100% sequence conservation between rrn leader regions of different species of mycobacteria, suggesting an essential function, whereas the boxB element, comprising a stem-loop structure, has no strict sequence conservation between species or between rrn operons.…”

mentioning

confidence: 99%

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

Arnvig¹,

Pennell²,

Gopal³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial NusA protein enhances transcriptional pausing and termination and is known to play an essential role in antitermination. Antitermination is signaled by a nut-like cis-acting RNA sequence comprising boxB, boxA, and boxC. In the present study, we demonstrate a direct, specific high-affinity interaction between the rrn leader nut-like sites and the NusA proteins of Mycobacterium tuberculosis and Escherichia coli. This NusA-RNA interaction relies on the conserved region downstream of boxA, the boxC region, thus demonstrating a key function of this element. We have established an in vivo assay for antitermination in mycobacteria and use this to show that the M. tuberculosis rrn nut-like site enhances transcriptional read-through of untranslated RNA consistent with an antitermination signal within this site. Finally, we present evidence that this NusA-RNA interaction affects transcriptional events further downstream.N usA (N-using substance A) is a highly conserved transcription factor involved in transcriptional pausing, termination, and antitermination (reviewed in ref. 1). The protein is also thought to be essential for the increased elongation rate associated with rrn transcription and to be a constituent of the rrn antitermination complex (2, 3).The rrn antitermination complex ensures that transcription of rRNA is not terminated by Rho despite the nascent transcripts being untranslated and thus naked (4). The complex is believed to include RNA polymerase (RNAP), Nus factors A, B, E, and G, ribosomal protein S4, and possibly one or more additional cellular components (5-10). The antitermination complex is assembled after transcription of a nut-like site (hereafter referred to as nut site for brevity) consisting of boxB, boxA, and boxC within the rrn leader region (1, 4). However, it has been shown that boxA is sufficient to obtain antitermination (7, 11). It is not entirely clear how the assembly proceeds, but evidence suggests that binding of a NusB͞NusE heterodimer to boxA RNA may play a part, whereas the exact roles of NusA, boxB, and boxC remain unclear (6,7,11,12).Bacterial NusA proteins harbor three RNA-binding domains: S1, K homology 1 (KH1), and KH2 as well as an N-terminal domain that interacts with the RNAP (13-15). KH domains, like ribosomal protein S1 domains, are found in a variety of nucleic acid-binding proteins, often in multiple copies within one protein (16). Structural evidence suggests that KH domains interact sequence-specifically with four to five consecutive nucleotides in single-stranded regions (17)(18)(19)(20). Comparable structures of S1 domains in complex with nucleic acids are not available. Based on the NusA structure of Thermotoga maritima, Worbs et al. (15) proposed that the three RNA-binding domains in the NusA protein act together to form an ''extended RNA binding surface'' consistent with the ability to bind RNA with high affinity and specificity. NusA interacts with boxAB (21) as well as with mRNA (22). Although bacterial NusA proteins display a high degree of co...

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“…The nucleotide sequence encoding the 16S rRNA gene accession no. X52927 (Kempsell et al, 1992) was analysed and primers were designed to amplify a fragment of 471 bp. The primers were designated 16SRibA (5'-CTGGCGGCGTGCTTAACACA-3') and 16SRibB (5'-ACGTAGTTGGCCGGTGCTTC-3') for the upstream and downstream primers, respectively.…”

Section: Rt-pcr For 16s Gene In M Tuberculosismentioning

confidence: 99%

The Mycobacterium tuberculosis homologue of the Mycobacterium avium mig gene is not specifically expressed in the macrophage

Alli¹,

Wilson²,

Ogbolu³

et al. 2010

African Journal of Biomedical Research

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With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of Mycobacterium avium was searched for in the M. tuberculosis database at The Institute of Genomic Research (TIGR), USA and The Sanger Institute, UK. Homologue of the gene was found and comprehensively analysed. Reverse transcription PCR (RT-PCR) was carried out on the mig (fadD19) gene homologue and echA19 gene. The result of the RT-PCR showed that the mig gene was at least 2-fold upregulated during intracellular infection of macrophage compared to the broth grown bacilli as opposed to the demonstrated specific expression of mig gene in M. avium infected macrophage. The echA19 gene was also found to be upregulated. .

show abstract

The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

Cited by 88 publications

References 35 publications

Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis The EMBL accession number for the sequence in this paper is X96711.

Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis The EMBL accession number for the sequence in this paper is X96711.

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

The Mycobacterium tuberculosis homologue of the Mycobacterium avium mig gene is not specifically expressed in the macrophage

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