Cloning and characterization in Escherichia coli of the gene encoding the principal sigma factor of an extreme thermophile, Thermus thermophilus.
The nucleotide sequence of the upstream region of the aspartate kinase genes of Thermus thermophilus HB27 revealed the presence of two open reading frames in the orientation opposite to that of the aspartate kinase genes. The upstream open reading frame termed ORF375 encodes a protein composed of 375 amino acid residues, possessing amino acid sequence motifs for methylases. Another open reading frame designated as sigA encodes a protein of 423 amino acid residues which shows significant identity in amino acid sequence to the principal sigma factor, a component of the DNA-dependent RNA polymerase holoenzyme. The close proximity of the open reading frames suggested that the two genes are transcribed in a polycistronic manner. By the use of an Escherichia coli expression system, SigA was produced in a soluble form. An in vitro transcription assay of purified SigA reconstituted with the core RNA polymerase of E. coli showed that Thermus SigA functioned as a sigma factor to initiate specific transcription.
Nishiyama
M
M
Biotechnology Research Center, University of Tokyo, Japan.
Kobashi
N
N
Tanaka
K
K
Takahashi
H
H
Tanokura
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012808:Sigma Factor / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases