Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli

Abstract

The complete nucleotide sequence of a 3541-base pairs (bp) DNA fragment from Bacillus stearothermophilus able to complement an Escherichia coli glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mutant ( gapD − ) has been determined. The B. stearothermophilus gap gene consists of a 1005-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Upstream from the start codon is a strong Shine-Dalgarno sequence typical of Gram-positive bacteria. Only one putative RNA polymerase recognition signal (−35 and −10 regions) is found 1153 bp upstream from the ATG start codon. In vivo utilization of this signal is in agreement with the study of gene expression from different subclones of the original fragment. 57 bp downstream from the TAA stop codon is a structure resembling Rho-independent transcription termination signals. Although B. stearothermophilus GAPDH-coding gene is highly expressed in E. coli, it contains several rare codons for E. coli. The predicted amino acid sequence of the GAPDH enzyme presents several differences with the amino acid sequence previously determined from the protein and is in better agreement with published crystallographic data.