RNA polymerase is an enzyme that catalyzes the synthesis of RNA from ribonucleoside triphosphates in the presence of a DNA template and divalent cation, such as Mg2+ or Mn2+. The RNA molecules are synthesized complementary and antiparalel to one of the DNA strands in a 5, to 3, direction. The ribonucleotides are covalently joined together by internucleoside 3, to 5, phosphodiester bonds with concomitant release of inorganic pyrophosphate.

RNA polymerases are large multiprotein complexes that range in size from 100 kDa (kilo Daltons) in T7 bacteriophage, to 400 kDa in bacteria and 500 kDa in eukaryotes. The bacterial RNA polymerase enzyme copies DNA into RNA at about 50 bases per second and makes a mistake in only about 1 in 10000 bases added. A proofreading mechanism is built into the enzyme complex, allowing it to detect and remove an incorrectly added nucleotide.

In order to perform its function, RNA polymerase enzyme must conduct these following steps:

• RNA polymerase must recognize the beginning of the DNA sequence to be transcribed within the double stranded template.
• Insert the correct nucleotide residue into each position, as determined by the DNA template sequence.
• Carry out synthesis, so that RNA is synthesized from beginning to end as a consequence of one polymerase binding event.
• Recognize the termination signals in order to the synthesis of RNA at the appropriate point.
• In order to conduct all of the steps above, the RNA polymerase should be able to recognize regulatory sites on the DNA template and interact with protein factors that modulate the activity of the enzyme.

The affinity of RNA polymerase for DNA is the result of electrostatic attractions, but is nonspecific. In E.coli, it is the sigma factor that ensures correct transcription initiation involving the stable binding of the RNA polymerase to the promoter. While in Eukaryotes, however, contain multiple DNA-dependent RNA polymerases. Those are RNA polymerases I, II, and III transcribing rRNA, mRNA and tRNA, and 5SrRNA, respectively.

RNA polymerase I synthesizes the eukaryotic rRNAs, 5.8S, 18S and 28S but not the 5S rRNA. RNA polymerases can transcribe a gene one following right after another. This high level of rRNA production allows the cell to respond rapidly to the need for new proteins by making the numerous ribosomes necessary to do the protein synthesis work. RNA polymerase II synthesizes mRNA from all genes encoding proteins, and also makes many ncRNAs. RNA polymerase III transcribes the transfer RNAs, the 5S rRNA needed for ribosomes and some of the snRNAs. RNA polymerase II transcribes more protein coding genes than RNA polymerase I or III.