DNA sequence and transcriptional characterization of a β-glucanase gene (celB) fromRuminococcus flavefaciensFD-1

1995 ◽  
Vol 41 (10) ◽  
pp. 869-876 ◽  
Author(s):  
Philip E. Vercoe ◽  
Jennie L. Finks ◽  
Bryan A. White
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Primer Extension ◽  
Start Site ◽  
Translation Start Site ◽  
Ruminococcus Flavefaciens ◽  
Endoglucanase Activity ◽  
Hydrophobic Cluster Analysis ◽  
E Coli ◽  
Translation Start

The recombinant clone pBAW101 (in pBluescript SK–) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69 414. A putative Shine–Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published β-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated from R. flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104. Transcription is initiated at different sites in E. coli and R. flavefaciens. In the case of R. flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resembling E. coli σ70-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA from E. coli was used in the primer extension analyses.Key words: Ruminococcus flavefaciens, endoglucanase, transcription, family 44 endoglucanase.

Nucleotide sequence and transcriptional analysis of the celD β-glucanase gene from Ruminococcus flavefaciens FD-1

1995 ◽  
Vol 41 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Philip E. Vercoe ◽  
Donn H. Spight ◽  
Bryan A. White
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Start Site ◽  
Ruminococcus Flavefaciens ◽  
E Coli ◽  
Translational Start Site ◽  
Translational Start

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44 631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine–Dalgamo-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a σ70-like −10 promoter sequence is present upstream from the transcription initiation site but there is no apparent −35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a σ70-like −10 region is present 5 bp upstream of this residue. Assuming 17 bp is the optimal distance between −10 and −35 sites for σ70 consensus sequences, the −35 region for celD transcription initiation in E. coli would be outside the boundaries of the cloned R. flavefaciens DNA.Key words: endoglucanase, xylanase, DNA sequencing, family E cellulase.

scholarly journals Characterization of the regulatory regions in the human desmoglein genes encoding the pemphigus foliaceous and pemphigus vulgaris antigens

1998 ◽  
Vol 329 (1) ◽  
pp. 165-174 ◽  
Author(s):  
J. Michael ADAMS ◽  
B. Martin REICHEL ◽  
A. Ian KING ◽  
D. Mark MARSDEN ◽  
D. Matthew GREENWOOD ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Pemphigus Vulgaris ◽  
Upstream Region ◽  
Lacz Gene ◽  
Start Site ◽  
Translation Start Site ◽  
Translation Start ◽  
Adhesive Proteins ◽  
Type 3

The adhesive proteins in the desmosome type of cell junction consist of two members of the cadherin superfamily, the desmogleins and desmocollins. Both desmogleins and desmocollins occur as at least three different isoforms with various patterns of expression. The molecular mechanisms controlling the differential expression of the desmosomal cadherin isoforms are not yet known. We have begun an investigation of desmoglein gene expression by cloning and analysing the promoters of the human genes coding for the type 1 and type 3 desmogleins (DSG1 and DSG3). The type 1 isoform is restricted to the suprabasal layers of the epidermis and is the autoantigen in the autoimmune blistering skin disease pemphigus foliaceous. The type 3 desmoglein isoform is also expressed in the epidermis, but in lower layers than the type 1 isoform, and is the autoantigen in pemphigus vulgaris. Phage ƛ genomic clones were obtained containing 4.2 kb upstream of the translation start site of DSG1 and 517 bp upstream of the DSG3 start site. Sequencing of 660 bp upstream of DSG1 and 517 bp upstream of DSG3 revealed that there was no obvious TATA box, but a possible CAAT box was present at -238 in DSG1 and at -193 in DSG3 relative to the translation start site. Primer extension analysis and RNase protection experiments revealed four putative transcription initiation sites for DSG1 at positions -163, -151, -148 and -141, and seven closely linked sites for DSG3, the longest being at -140 relative to the translation start site. The sequences at these possible sites at -166 to -159 in DSG1 (TTCAGTCC) and at -124 to -117 in DSG3 (CTTAGACT) have some similarity to the initiator sequence (CTCANTCT) described for a TATA-less promoter often from -3 to +5, and the true transcription initiator site might therefore be the A residue in these sequences. There were two regions of similarity between the DSG1 and DSG3 promoters just upstream of the transcription initiation sites, of 20 and 13 bp, separated by 41 bp in DSG1 and 36 bp in DSG3. The significance of these regions of similarity remains to be elucidated, but the results suggest that they represent a point at which these two desmoglein genes are co-ordinately regulated. Analysis of the upstream sequences revealed GC-rich regions and consensus binding sites for transcription factors including AP-1 and AP-2. Exon boundaries were conserved compared with the classical cadherin E-cadherin, but the equivalent of the second cadherin intron was lacking. A 4.2 kb region of the human DSG1 promoter sequence was linked to the lacZ gene reporter gene in such a way that there was only one translation start site, and this construct was used to generate transgenic mice. We present the first transgenic analysis of a promoter region taken from a desmosomal cadherin gene. Our results suggest that the 4.2 kb upstream region of DSG1 does not contain all the regulatory elements necessary for correct expression of this gene but might have elements that regulate activity during hair growth.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

49: Discovery of SMAD4 Promoter 16 Kb Upstream From its Translation Start Site

2009 ◽  
Vol 151 (2) ◽  
pp. 193 ◽  
Author(s):  
D. Calva ◽  
S. Chinnathambi ◽  
J.R. Howe
Keyword(s):  
Start Site ◽  
Translation Start Site ◽  
Translation Start

scholarly journals The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site

2021 ◽  
Vol 12 ◽  
Author(s):  
Christian Otten ◽  
Tanja Seifert ◽  
Jens Hausner ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Structural Component ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Start Site ◽  
Translation Start Site ◽  
Terminal Protein ◽  
Type Iii ◽  
Translation Start ◽  
Membrane Spanning

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Determination of the translation start site of the large subunit of ribulose-1,5-bisphosphate carboxylase from maize

1984 ◽  
Vol 3 (6) ◽  
pp. 403-406 ◽  
Author(s):  
Mark Bloom ◽  
Nathan Brot ◽  
Bennett N. Cohen ◽  
Herbert Weissbach
Keyword(s):  
Large Subunit ◽  
Start Site ◽  
Translation Start Site ◽  
Bisphosphate Carboxylase ◽  
Translation Start
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