Gene, 119 (1992) 147-148 0 1992 Elsevier Science Publishers
B.V. All rights reserved.
amino acid sequence;
and Jozef Sevcik
Institute of Molecular Biology, Slovak Academy of Sciences, 842 51 Bratislava. Czechoslovakia Received
by C.R. Hutchinson:
April 1992; Received
11 June 1992
A ribonuclease-encoding gene (rnaSa3) from Streptomyces aureofaciens CCM3239 has been isolated deduced amino acid sequence shows 77% homology with RNase Sa from S. aureofaciens.
gene was isolated from S. aureofaciens genomic library by hybridization of recombinant colonies with a 17-mer oligo probe 5’-GTGTAGTAGTCCTCCTG-3’ homologous to the -Gln77-G1u78Asp79-Tyr80-Tyr81- of the RNase Sa aa sequence (Shlyapnikov et al., 1986). The complete nt sequence of rnaSa3 is shown in Fig. 1. Starting with the ATG codon at nt 196, there is an ORF of 423 bp terminating with TGA at nt 6 19. The initial Met is followed by a leader sequence with five possible sites where the signal peptide could be cleaved. These sites conform to the (-3, -1) rules of von Heijne (1986). The stop codon is followed by a region where a possible RNA stem-loop structure may be found. The N-terminal aa was identified on the basis of the known RNase Sa aa sequence and will be verified after isolation and sequencing of the enzyme from S. aureofaciens CCM3239 strain. The
Correspondence to; Dr. J. Sevcik, Institute Academy
vakia. Tel. (42-7) 376556; Abbreviations: RNase
842 51 Bratislava,
Fax (42-7) 372316.
aa, amino acid(s); bp, base pair(s); nt, nucleotide(s);
Sa3; S., Streptomyces.
maSa3, gene en-
RNase Sa3 belongs to the microbial RNase family, which is divided into fungal and bacterial subgroups. The moststudied is RNase Tl secreted by the fungus Aspergillus oryzae (Pace et al., 1991). The homology between the deduced aa sequence of RNase Sa3 and the aa sequence of RNase Tl is very low (17%). RNase Sa3 has the highest homology (77%) with RNase Sa. The aa sequences of RNase Sa3, RNase Tl and RNase Sa are shown in Fig. 2. Five of the aa, Asn39, G1u41, GAUGE,Arg69, and His85 (numbered according to RNase Sa) are conserved in all microbial RNases. Based on the known tertiary structure of RNase Sa (Sevcik et al., 1988; 1991), it appears that all aa residues which differ between RNase Sa3 and RNase Sa are localized at the surface of the molecule. It is expected that RNase Sa3 has an almost identical tertiary structure and similar enzymatic properties to RNase Sa. This study is currently in progress. These studies were supported by a Grant GA138/91 from the Slovak Academy of Sciences. The authors thank Dr. I. Rybajlak for synthesizing the 17-mer oligo probe and to the Czechoslovak Collection of Microorganisms (CCM) for providing us with the strain S. aureofaciens CCM3239.
DVSG~~~CLSALPPEATDTL-NLIA~~PFPY~QDG~=Q~~~VLPT*~YG .. . .... :: :::: : ::: :: :: KAVGRVCYSALPSQAHDTL-DLIDEGGPFPYSQDGVVFQNREGLLPAHSTG
YYH~YT"~TPGARTRGT----RBIICGEATQRDYYTGD~AT~SLIDQTC ::: : :::::: :::: : YYHEYTVITPGSP~GA----RRIITGQQWQEDYYTADHYASFBFA~
PYYEWP~LSS&YS~PGSGADkVFNRN-NQLAGV~Ti3TGASGNNF"R;T Ip t +
Fig. 2. Comparison
the aa sequences 301
Pace, C.N., Heinemann, structure,
by the method
sion No.: M82920.
where the signal peptide could be cleaved.
The gene was se-
DNA with base
65 (1980) 499-560.
U., Hahn, E. and Saenger, W.: Ribonuclease
G.G. and Zelinka,
Int. Ed. Engl. 30 J.: The X-ray anal-
1988, pp. 9-18.
E.J. and Dodson,
of the crystal structure
its complex with 3’guanylic
with five possible
least squares refinement
sequence of the maSa3 gene, and deduced
Sevcik, J., Dodson,
Sac11 CCGTCCGGGGCGGGACGGCGCCTCGTCCG~C~CCGCTACGAG~CTGGAGGA -Xc---SacII CGGAGAGGATGTTGCC?3Z%GGATCGGTGAACAGGCGATCAGCG
and those con-
ysis of ribonuclease Sa. In: Zelinka, J. and Balan, J. (Eds.), Metabolism and Enzymology of Nucleic Acids Including Gene Manipu-
Ta I CGGG ld ACTTCGCCTGCTGATCCCOOCTCAGCCCCCCACCCACGCCGCCGTCCCCC~~C xxi-xRVDFAC
by a leader sequence
(1991) 343-360. Sevcik, J., Dodson, E.J., Dodson,
A.M. and Gilbert,
The initial Met is followed
Sa3 (Fig. 1) with
et al., 1986) and RNase Tl
Fig. 1. Nucleotide
EcoRII TCC~ACDGCGTCGTCTTCCAGAI\CCGCGAOOOCCTGCTGCCCGCCCACAGCAC~GC TthlllI SQDGVVFQNREGLLPARSTG
(Takahashi, 1985). Identical aa are indicated by colons, served in all microbial RNases are marked by arrows.
of deduced of RNase
acid at 1.8 A resolution.
(1991) 240-253. Shlyapnikov, S.V., Both, V., Kulikov,
Acta Cryst. B47 A.A., Sevcik, J.
and Zelinka, J.: Amino acid sequence determination
(1986) 335-339. Takahashi, K.: A revision of ribonuclease von Heijne,
G.: A new method
of the amino acid sequence
98 (1985) 815-817. for predicting
sites. Nucleic Acids Res. 14 (1986) 4683-4690.