OVERPRODUCTION OF SAC7D AND SAC7E REVEALS ONLY SAC7E TO BE A DNA-BINDING PROTEIN WITH RIBONUCLEASE-ACTIVITY FROM THE EXTREMOPHILIC ARCHAEONSULFOLOBUS-ACIDOCALDARIUS

Genomic DNA from Sulfolobus acidocaldarius was screened using a degene rate oligodeoxyribonucleotide, derived from the sequence of 16 N-termi nal amino acids from SaRD protein. SaRD protein was previously isolate d in our laboratory and identified as a protein from S. acidocaldarius exhibiting ribonuclease activity as well as DNA-binding properties. O n the basis of Southern hybridization analysis two genes from S. acido caldarius have been cloned, sequenced and over-produced 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 Sac7 e 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 exh ibits RNase activity and is catalytically indistinguishable from SaRD protein. Western blot analysis using a polyclonal antiserum raised aga inst purified SaRD protein further confirmed that Sac7e and SaRD are i dentical proteins endowed with RNase activity and DNA-binding properti es. 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 clos ely related 7 kDa proteins from two Sulfolobus strains converting DNA- binding proteins into RNases are pointed out and discussed, whereas su bstitutions of Glu by Gin (S. solfataricus) or by Lys (S. acidocaldari us) seem to be crucial.