| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
UP TO 10 POLYPEPTIDES ASSOCIATED WITH RNA POLYMERASE AND WHICH MAY HAVE SOME FUNCTIONS MODULATING RNA POLYMERASE DURING TRANSCRIPTION HAVE BEEN IDENTIFIED IN THIS LABORATORY. STRINGENT STARVATION PROTEIN (SSP) IS ONE SUCH POLYPEPTIDE. TO STUDY THE PHYSIOLOGICAL FUNCTION OF SSP, WE CLONED THE GENE FOR THE PROTEIN, AND THE NUCLEOTIDE SEQUENCE OF IT WAS DETERMINED. THE DEDUCED AMINO ACID SEQUENCE SHOWS THAT SSP IS COMPOSED OF 212 AMINO ACID RESIDUES WITH A MOLECULAR WEIGHT OF 24,305. WE IDENTIFIED A SITE ON THE CHROMOSOME DNA CORRESPONDING TO THE 5' END OF THE IN VIVO TRANSCRIPT OF THE SSP GENE. HOWEVER, THE CONSENSUS PROMOTER SEQUENCES WERE NOT FOUND AT THE UPSTREAM REGION. IN THE 3' FLANKING REGION A LONG CODING FRAME WAS FOUND IMMEDIATELY FOLLOWING THE SSP GENE, SUGGESTING THAT THE SSP GENE IS A MEMBER OF A MULTICISTRONIC OPERON. TO IDENTIFY THE PROMOTER SIGNALS OF THE GENE, DELETION MAPPING OF THE UPSTREAM REGION OF THE STRUCTURAL GENE WAS PERFORMED. LIKE ORDINARY PROMOTERS OF E. COLI, THE UPSTREAM 40 BP FROM THE TRANSCRIPTION INITIATION SITE WAS REQUIRED. IN ADDITION, THE PRESENCE OF FURTHER UPSTREAM 40 BP STIMULATES THE TRANSCRIPTION SEVERAL FOLDS. THIS CIS-ACTING REGION CONTAINS STRETCHES OF PYRIMIDINE CLUSTERS AND EXHIBITS AB=NORMAL MOBILITY ON POLYACRYLAMIDE GEL ELECTROPHORESIS.
A PLASMID CARRYING THE SSP GENE WAS INTEGRATED AT THE CHROMOSOMAL SSP GENE OF A polA STRAIN, AND THE AMPICILLIN-RESISTANT GENE OF THE PLASMID WAS USED AS A MARKER TO DETERMINE THE MAP POSITION OF THE SSP GENE. IT WAS MAPPED TO THE REGION BETWEEN gltB AND glnF AT MIN 69.5. IT WAS THUS ES=TABLISHED THAT THE SSP GENE IS A HITHERTO UNKNOWN GENE. CONSTRUCTING VARIOUS STRAINS DELETING THE SSP GENE, WE ARE NOW TRYING TO DETERMINE WHETHER SSP IS ESSENTIAL FOR CELL GROWTH OR NOT. AT LEAST IT DOES NOT SEEM TO BE ESSENTIAL FOR CELLS GROWING UNDER ORDINARY CONDITIONS.
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