Overproduction of Sac7d and Sac7e reveals only Sac7e to be a DNA-binding protein with ribonuclease activity from the extremophilic archaeon Sulfolobus acidocaldarius.
Genomic DNA from Sulfolobus acidocaldarius was screened using a degenerate oligodeoxyribonucleotide, derived from the sequence of 16 N-terminal amino acids from SaRD protein. SaRD protein was previously isolated in our laboratory and identified as a protein from S. acidocaldarius exhibiting ribonuclease activity as well as DNA-binding properties. On the basis of Southern hybridization analysis two genes from S. acidocaldarius have been cloned, sequenced and overproduced in Escherichia coli. The deduced amino acid sequences revealed that one gene encodes Sac7d and the other one Sac7e; two small, previously described basic proteins from S. acidocaldarius, and furthermore the N-termini of Sac7e and SaRD are identical. Northern blot analysis demonstrated that the genes are transcribed separately. After expression of sac7d and sac7e genes in E. coli it was shown that only recombinant Sac7e protein exhibits RNase activity and is catalytically indistinguishable from SaRD protein. Western blot analysis using a polyclonal antiserum raised against purified SaRD protein further confirmed that Sac7e and SaRD are identical proteins endowed with RNase activity and DNA-binding properties. A new RNA cleavage mechanism has to be postulated for Sac7e since, in contrast to common RNases (e.g. RNase A and T1), no histidines are present in the amino acid sequence. Differences between the very closely related 7 kDa proteins from two Sulfolobus strains converting DNA-binding proteins into RNases are pointed out and discussed, whereas substitutions of Glu by Gln (S. solfataricus) or by Lys (S. acidocaldarius) seem to be crucial.
Kulms
D
D
Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Germany.
Schäfer
G
G
Hahn
U
U
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D012260:Ribonucleases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D016945:Sulfolobus acidocaldarius / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C095116:Sac7 protein, Sulfolobus; D012260:Ribonucleases