Regulation of glycine tRNA gene expression in the posterior silk glands of the silkworm Bombyx mori.

Abstract

The glycine tRNA genes in silkworm Bombyx mon contain two regulatory regions upstream of the transcription start site as identified by direct transcription of 5' deletion mutants, transcription competition, gel mobility shift assays, and footprinting. A positive regulatory region is present in the immediate 5' flanking sequences of the four tRNAGIY clones studied. This region is essential for cell-free transcription in homologous extracts. A negative regulatory region is present farther upstream, and transcription competition experiments indicate its presence in three of the four clones exmned.The posterior silk gland of the silkworm Bombyx mori is a highly specialized organ involved in the synthesis of enormous amounts of the silk protein fibroin. Fibroin is rich in glycine (43%), alanine (28%), serine (12%), and tyrosine (5%) (1). To gear the system for synthesis of large quantities of fibroin, there is a functional adaptation in the silk glands of the B. mori larvae (2). The tRNA population in the posterior silk gland becomes highly enriched for the tRNA species cognate to the predominant amino acids in fibroin (3-5). To understand how the posterior silk gland produces such high levels of defined tRNA species, we have undertaken the study of regulation of expression of glycine tRNA genes. No tRNAGlY species that are exclusive to the silk gland have been identified so far (6-8), unlike the silk gland-specific tRNAAla (9, 10). It was therefore important to compare the relative transcriptional efficiencies of more than one tRNAly gene. In this paper, we report the presence of two regulatory regions for these tRNA GlY genes. An upstream positive regulatory region, within 40 base pairs (bp) of the transcription start site, is required for cell-free transcription of the clones studied. A negative regulatory element is present further upstream in three of the four clones examined. MATERIALS AND METHODSConstruction of Deletion Mutants. The six tRNAGly genes used in this study were a gift from A. Fournier. These clones were isolated from a B. mori genomic library in the phage A vector Charon 4. We have subcloned one of these, pBma2 (8) as an EcoRI-Sau3Al fragment in pUC18, which was designated pR8. The deletion mutants were constructed from this subclone. The 5' deletion constructs include pX3 (-2 to +108), pSX (-40 to +108), and pKX (-150 to +108). The subclone pA3 has all of the upstream sequences of pR8 (-300) and the coding region up to the Sma I site (+53). These constructs are listed in Fig. 1B.Preparation of Nuclear Extracts and in Vitro Transcription. Nuclear extracts from posterior silk glands of the fifth instar larvae were prepared as described for HeLa cell extracts (11) with modifications. The glands were homogenized in a buffer containing 2 M sucrose, 10o (vol/vol) glycerol, 10 mM Hepes (pH 7.9), 15 mM KCI, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was layered on a 3-ml cushion of the same buffer and centrifuged at 26,000 rpm for 1 hr at 40C in a Beckman SW-4...

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“…Nuclear extracts from posterior silk glands of B. mori in the fifth larval instar were prepared as described previously [16]. Briefly, the silk glands (freshly dissected or glands frozen at )70°C up to 6 months) were homogenized (Dounce homogenizer) in 10 mM Hepes (pH 7.9), containing 2 M sucrose, 10% glycerol, 15 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 0.15 mM spermine, 0.15 mM spermidine and 1 mM EDTA.…”