Multisubunit RNA polymerases (RNAPs) are complex molecular machines that are responsible for transcription of genes in all cellular organisms and possess several catalytic activities, the most important of which are nucleotide addition to the growing RNA chain and RNA cleavage. Switching between different RNAP activities is essential for efficient and accurate RNA synthesis and depends on the structural state of the elongation complex and the conformational dynamics of the active center of the enzyme. The active center of RNAP contains two magnesium ions, which coordinate the reactive groups of substrates, and structural elements that are involved in the binding and correct orientation of substrates and in RNAP translocation. The most important of these elements are G-loop, F-helix, and regions that affect their conformational mobility. The review discusses the mechanisms of structural rearrangements that take place in the active center of RNAP during transcription. Several examples are provided for the RNAP regulation by factors that affect the binding of the catalytic magnesium ions and the conformational mobility of the G-loop and F-helix.