It is necessary to know the concentration of free intracellular RNA polymerase to design meaningful in vitro transcription experiments. One method of determining the concentration utilizes the fact that the β and β’ subunits of the E. coli RNA polymerase are larger than most other polypeptides in the cell. This permits them to be easily separated from other cellular proteins by SDS polyacrylamide gel electrophoresis. Consequently, after such electrophoresis, the amount of protein in the β and β’ bands is compared to the total amount of protein on the gel. The results are that a bacterial cell contains about 3,000 molecules of RNA polymerase. A calculation using the cell doubling time and amounts of messenger RNA, tRNA, and ribosomal RNA in a cell leads to the conclusion that about 1,500 RNA molecules are being synthesized at any instant. Hence half the cell’s RNA polymerase molecules are synthesizing RNA. Of the other 1,500 RNA polymerase molecules, fewer than 300 are free of DNA and are able to diffuse through the cytoplasm. The remainder are temporarily bound to DNA at nonpromoter sites.

How do we know these numbers? On first consideration, a direct physical measurement showing that 300 RNA polymerase molecules are free in the cytoplasm seems impossible. The existence, however, of a special cell division mutant of E. coli makes this measurement straight forward (Fig. 1). About once per normal division, these mutant cells divide near the end of the cell and produce a minicell that lacks DNA. This cell contains a sample of the cytoplasm present in normal cells. Hence, to determine the concentration of RNA polymerase free of DNA in cells, it is necessary only to determine the concentration of ββ’ in the DNA-less minicells. Such measurements show that the ratio of ββ’ to total protein has a value in minicells of one-sixth the value found in whole cells. Thus, at any instant, less than 20% of the RNA polymerase in a cell is free within the cytoplasm.

Fig1. The generation of a minicell lacking a chromosome.

مواضيع ذات صلة

Measuring the Activity of RNA Polymerase

Use of Transgenic and Knockout/Knockin Organisms to Model Gene Function

The Polymerase Chain Reaction

الإضعاف :التوقف المبكر للاستنساخ (Attenuation: the premature termination of transcription)

كابح التربتوفان tryptophan repressor

Gel Electrophoresis Assay of Eukaryotic Replication Origins

Constancy of the E. coli DNA Elongation Rate

Bidirectional Replication from E. coli Origins

أوبرون اللاكتوز lac operon

اكتشاف الأوبرون

مثبطات تخليق البروتين .... وسيلة للعلاج

تحوير سلاسل الأحماض الأمينية الجانبية للبروتين (Amino acids side chains modifications)