Cloning and sequencing of the gene encoding a ribonuclease from Streptomyces aureofaciens CCM3239

Cloning and sequencing of the gene encoding a ribonuclease from Streptomyces aureofaciens CCM3239

Gene, 119 (1992) 147-148 0 1992 Elsevier Science Publishers GENE B.V. All rights reserved. 147 0378-l 119/92/$05.00 06659 Cloning and sequenci...

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Gene, 119 (1992) 147-148 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

147

0378-l 119/92/$05.00

06659

Cloning

and

sequencing

Streptomyces (Recombinant

of

the

gene

encoding

a

ribonuclease

from

aureofaciens CCM3239

DNA;

RNase;

Dagmar Homerova,

oligodeoxyribonucleotide

Zuzana

Holhderova,

probe; deduced

Jan Kormanec

amino acid sequence;

nucleotide

sequence)

and Jozef Sevcik

Institute of Molecular Biology, Slovak Academy of Sciences, 842 51 Bratislava. Czechoslovakia Received

by C.R. Hutchinson:

30 December

1991; Revised/Accepted:

3 April/l3

April 1992; Received

at publishers:

11 June 1992

SUMMARY

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.

rnaSa3

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

CCM3239

Correspondence to; Dr. J. Sevcik, Institute Academy

of Sciences,

Dubravska

vakia. Tel. (42-7) 376556; Abbreviations: RNase

Biology,

842 51 Bratislava,

Slovak

Czechoslo-

Fax (42-7) 372316.

aa, amino acid(s); bp, base pair(s); nt, nucleotide(s);

ohgodeoxyribonucleotide; coding

cesta,

of Molecular

ORF,

open reading

Sa3; S., Streptomyces.

frame;

oligo,

maSa3, gene en-

and sequenced.

The

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.

148

121

CCGGGCCGTGAACAGGCAAGAAATCAACGTAGTTGACGTTGACGGCCGCAGCTCTACGCG

181

,,,,A=

RN&se

sa

RNase

sax

RNa8e

Sa

RNase

Sa3

RNase

Tl

DVSG~~~CLSALPPEATDTL-NLIA~~PFPY~QDG~=Q~~~VLPT*~YG .. . .... :: :::: : ::: :: :: KAVGRVCYSALPSQAHDTL-DLIDEGGPFPYSQDGVVFQNREGLLPAHSTG

YYH~YT"~TPGARTRGT----RBIICGEATQRDYYTGD~AT~SLIDQTC ::: : :::::: :::: : YYHEYTVITPGSP~GA----RRIITGQQWQEDYYTADHYASFBFA~

NarI

GACGGCATGAGAATCCCCCCTCGCCTCGTCGCCCTCGCGW-GCAGCC

: :

:

MRIPPRLVALAGAAA

:

PYYEWP~LSS&YS~PGSGADkVFNRN-NQLAGV~Ti3TGASGNNF"R;T Ip t +

241

Fig. 2. Comparison

16

the aa sequences 301

361

AtA

76

481

136

661

721

A*S

A+S

"

C 2%

K

A

"

G

R

"

C

Y

S

A

NarI TACTACCACGAGTACACCGTCATCACCCCCGGCTCGCCCACGCGCG~CGCCGGATC

Pace, C.N., Heinemann, structure,

ITGQQWQEDYYTADHYASFR

quenced

indicate

lation

by the method

sion No.: M82920.

aa sequence.

where the signal peptide could be cleaved.

a possible

RNA

of Maxam

stem-loop

structure.

and Gilbert

posiFacing

The gene was se-

(1980). GenBank

end-labeled

Enzymol.

DNA with base

65 (1980) 499-560.

U., Hahn, E. and Saenger, W.: Ribonuclease

Plenum,

Angew.

Chem.

G.G. and Zelinka,

New York,

Tl:

Int. Ed. Engl. 30 J.: The X-ray anal-

acces-

1988, pp. 9-18.

E.J. and Dodson,

G.G.: Determination

of the crystal structure

its complex with 3’guanylic

with five possible

Methods

and stability.

least squares refinement

sequence of the maSa3 gene, and deduced

arrows)

function

Sevcik, J., Dodson,

Sac11 CCGTCCGGGGCGGGACGGCGCCTCGTCCG~C~CCGCTACGAG~CTGGAGGA -Xc---SacII CGGAGAGGATGTTGCC?3Z%GGATCGGTGAACAGGCGATCAGCG

arrows

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

W.: Sequencing

cleavage.

(1991) 343-360. Sevcik, J., Dodson, E.J., Dodson,

ATCACCGGTCAGCAGTGGCAGGAGGACTACTACACCGCCGACCACTACGCCTCCTT~CG~

tions (upward

A.M. and Gilbert,

specific chemical

YYHEYTVITPGSPTRGARRI

The initial Met is followed

Sa3 (Fig. 1) with

et al., 1986) and RNase Tl

REFERENCES Maxam,

Fig. 1. Nucleotide

of RNase

AVLII CCTCATAGACGAGGGCCGTTCCCGTAC

EcoRII TCC~ACDGCGTCGTCTTCCAGAI\CCGCGAOOOCCTGCTGCCCGCCCACAGCAC~GC TthlllI SQDGVVFQNREGLLPARSTG

116

601

A

LPSQAHDTLDLIDEGGPFPY

96

541

S

ErxJRlI CTGCCCTCm?%CGCACGACACCC

56

421

S

aa sequence

Sa (Shlyapnikov

(Takahashi, 1985). Identical aa are indicated by colons, served in all microbial RNases are marked by arrows.

GCAGCCTCCTCCGCCGCCTCCdCATCGGTGllAGGCGGTGACTCGGCG

36

of deduced of RNase

acid at 1.8 A resolution.

(1991) 240-253. Shlyapnikov, S.V., Both, V., Kulikov,

and restrained

of ribonuclease

V.A., Dementiev,

Sa and

Acta Cryst. B47 A.A., Sevcik, J.

and Zelinka, J.: Amino acid sequence determination

of guanyl-specific

ribonuclease

FEBS

Sa from

(1986) 335-339. Takahashi, K.: A revision of ribonuclease von Heijne,

Streptomyces

aureofaciens.

and confirmation

Tl. J.Biochem.

G.: A new method

Lett.

209

of the amino acid sequence

98 (1985) 815-817. for predicting

signal sequence

sites. Nucleic Acids Res. 14 (1986) 4683-4690.

cleaving