This work presents the results of single-molecular studies of the effect of magnesium ions on the dynamic characteristics of transcription elongation of bacterial RNA polymerase. It has been shown that the instantaneous and average transcription rates decrease with a decrease in magnesium concentration. The observed dependence occurred due to an increase in the number of short pauses; an explanation of the mechanism of their formation was put forward. To carry out these studies, the method of acoustic force spectroscopy (AFS) was used. This technique served as a basis for the development of a single-molecule procedure for characterizing the transcription parameters. A detailed description of the method and algorithm for processing the measurement results was given.