Transcriptional Analysis and Regulation of Expression of the Scr FI Restriction-Modification System of Lactococcus lactis subsp. cremoris UC503
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ABSTRACT Scr FI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame ( orfX ) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, and scrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5′ extension analysis indicated that the start site for the scrFIAM promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for the orfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M. Scr FIA). In order to determine if this motif played a role in regulation of the Scr FI locus, M. Scr FIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the Scr FI operon, one located upstream of orfX and the other located just upstream of scrFIAM . M. Scr FIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of the Scr FI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M. Scr FIA in regulation of both promoters on the Scr FI locus.Keywords:
Primer extension
Expression of the isiA and isiB genes was analysed in the cyanobacterium Synechocystis sp. PCC 6803 grown in high salt or in iron-deficient medium. The detection of a 2.3-knt transcript in Northern blot experiments indicated cotranscription of isiAB in an operon, which was confirmed by reverse transcriptase PCR. The abundance of a monocistronic 1.25-knt isiA-specific mRNA was about 10-fold higher than the dicistronic message. The isiAB-specific transcripts were most abundant in cells adapted to 342 mM NaCl and under iron deficiency. The promoter of the operon was mapped to 211 bp upstream of the translational start. A putative Fur binding site was detected immediately upstream of the GTG start codon. A preliminary transcript of about 0.2 knt was detected in cells grown in conditions in which the isiAB operon was not transcribed. This indicates that a repressor binds to the identified Fur binding site and thus inhibits isiAB transcription under low salt and iron replete conditions.
Synechocystis
Transcription
Northern blot
trp operon
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Primer extension
gal operon
trp operon
L-arabinose operon
Transcription
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DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.
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Hydrogenase
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gal operon
Transcription
trp operon
Ribosomal protein
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Summary The promoter region of the cryIIIA toxin gene of Bacilius thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions ‐635 to ‐553 (with reference to the translational start codon of cryIIIA) , an internal region extending from nucleotide positions ‐553 to ‐367, and a downstream region extending from nucleotide position 367 to +18. Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region. Primer extension experiments reveal a major cryIIIA transcript (designated T‐129) starting at nucleotide position ‐129 and another transcript (designated T‐558) starting at nucleotide position ‐558. Mutation in the ‐35 region of the promoter responsible for the initiation of T‐558 indicates that the upstream promoter is essential for full expression of cryIIIA , although not sufficient. Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5 end of T‐129. Taken together, these results indicate that the 5 end of T‐129 is not a transcriptional start site. Therefore, we propose that T‐129 results from the processing of the mRNA initiated at the upstream promoter (T‐558), generating a stable mRNA with a 5’extremity at nucleotide position ‐129. From primer extension analysis and transcriptional fusions to lacZ , it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporula‐tion and remains active at least until t 5 . However, unlike the promoters of other cry genes, this promoter is similar to σ a ‐dependent promoters rather than sporulation‐specific promoters. This promoter may therefore be transcribed by the Eσ A form of RNA polymerase. Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary‐phase‐specific activator.
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Expression of the isiA and isiB genes was analysed in the cyanobacterium Synechocystis sp. PCC 6803 grown in high salt or in iron-deficient medium. The detection of a 2.3-knt transcript in Northern blot experiments indicated cotranscription of isiAB in an operon, which was confirmed by reverse transcriptase PCR. The abundance of a monocistronic 1.25-knt isiA-specific mRNA was about 10-fold higher than the dicistronic message. The isiAB-specific transcripts were most abundant in cells adapted to 342 mM NaCl and under iron deficiency. The promoter of the operon was mapped to 211 bp upstream of the translational start. A putative Fur binding site was detected immediately upstream of the GTG start codon. A preliminary transcript of about 0.2 knt was detected in cells grown in conditions in which the isiAB operon was not transcribed. This indicates that a repressor binds to the identified Fur binding site and thus inhibits isiAB transcription under low salt and iron replete conditions.
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Transcription
Northern blot
trp operon
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Bacillus anthracis
Stop codon
Anthrax toxin
Reading frame
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We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.
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Shine-Dalgarno sequence
Ribosomal binding site
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The expression of the cydDC operon was investigated by using a chromosomal phi(cydD-lacZ) transcriptional fusion and primer extension analysis. A single transcriptional start site was found for cydD located 68 bp upstream of the translational start site, and Northern blot analysis confirmed that cydDC is transcribed as a polycistronic message independently of the upstream gene trxB. cydDC was highly expressed under aerobic growth conditions and during anaerobic growth with alternative electron acceptors. Aerobic expression was independent of ArcA and Fnr, but induction of cydDC by nitrate and nitrite was dependent on NarL and Fnr.
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Bacillus thuringiensis
Ribosomal binding site
Stop codon
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