Abstract
Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) was shown to occur mainly at the level of translation by measuring OppA synthesis and its mRNA level. Several artificial oppA genes were constructed by site-directed mutagenesis. These synthesize different kinds of OppA mRNAs: mRNAs differing in the size of 5'-untranslated region; mRNAs having the Shine-Dalgarno (SD) sequence in a different position; mRNAs having different secondary structure in the region of the SD sequence; and fusion mRNAs consisting of the 5'-untranslated region of OppA mRNA and the open reading frame of beta-galactosidase. By measuring the synthesis of OppA or beta-galactosidase from these mRNAs, we found that the 171-nucleotide 5'-untranslated region and 145 nucleotides of the ORF of OppA mRNA are involved in the polyamine stimulation of OppA synthesis. When the secondary structure of the above region of OppA mRNA was analyzed by optimal computer folding, it was shown that the degree of polyamine stimulation of OppA protein synthesis was dependent on the structure of the SD sequence in addition to its position. Loose base pairing of the SD sequence with other regions of the mRNA caused strong polyamine stimulation, while intense base pairing of the SD sequence with other regions of the mRNA resulted in insignificant or weak polyamine stimulation.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) D83137
We have shown that stimulation of OppA synthesis by polyamines occurs mainly at the level of translation, and the position and secondary structure of the Shine-Dalgarno (SD) sequence [14] are probably involved in the stimulation of protein synthesis by polyamines
When cells were transformed with pMW975, a low copy number plasmid, OppA synthesis was greatly stimulated by polyamines
Summary
Bacterial Strains and Culture Conditions—E. coli MA261 (speB speC gly leu thr thi) was kindly provided by Dr W. E. coli MA261 oppA::Km and lacZ::Em were prepared as described previously [15, 16] These cells were grown at 37 °C in medium A in either the presence (100 g/ml) or absence of putrescine [13]. Methionine content in medium A was decreased from 100 to 3 g/ml in order to label proteins with [35S]methionine; this modification did not influence their growth rate Another polyaminerequiring mutant, HT283, was kindly provided by Dr H. A PCR product was obtained using the HindIII-digested pMW975 as template, and 5Ј-CAAATAGGTTACCTGGT-3Ј and 5Ј-GGGGAATTCCAATCTCTATTTGATTGA-3Ј as primers. PMW412 was constructed by inserting the 1.1-kb EcoRI-BstEII fragment of the PCR product into the same restriction sites of pMW211. Two PCR products obtained were digested with EcoRI and BstEII, and inserted into the same restriction sites of pMW211. The same method was applied to prepare pMWSD1 to pMWSD5 with appropriate primers
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