The microcalorimetric method and DNA site-directed mutagenesis technique were used together to study the effect of transcription start site mutagenesis on RM07 promoter activity and gene transcription efficiency in Escherichia coli. The results revealed that once the putative transcription start site G was mutated to T, the promoter activity of RM07 promoter fragment was decreased and the transcription strength of cat reporter gene was weakened. Our work suggests that the nucleotide component of transcription start site is very critical for the promoter strength and the gene transcription efficiency. Our research also provides a new and useful technique for determining the transcription start site using both chemical and biological method.