Transcription requires an enzyme to catalyze the synthesis of the RNA molecule. The enzyme responsible for this is called RNA polymerase. (RNA polymerases are a class of enzyme, based on their function. We've seen a member of this class elsewhere - primase from DNA replication is an RNA polymerase. However, the enzyme for transcription is named 'RNA polymerase'.) RNA polymerase, when it was first isolated in the late 1950's, was found to be able to synthesize RNA in vitro, and to have some of the same requirements as DNA polymerase. For example, RNA polymerase required a DNA template. It also required nucleotide triphosphates (although in this case it was ribonucleotide triphosphates instead of deoxyribonucleotide triphosphates, as it was for DNA polymerase). (For more on the structure of nucleotides, see the module on nucleic acid structure.) One way that RNA polymerase differed from DNA polymerase was that RNA polymerase did not require a primer to initiate synthesis. RNA polymerase, just like DNA polymerase, synthesized only in the 5' to 3' direction.
Transcription in Prokaryotes
RNA polymerase in prokaryotes is made up of five subunits: two alpha subunits, one beta subunit, one beta prime subunit, and one sigma subunit. This configuration is called the RNA polymerase holoenzyme. The whole enzyme has a molecular weight of about 500,000. Of the various subunits, the beta and beta prime subunits are most important for catalytic activity; the sigma subunit is involved in regulation of transcription.
Transcription consists of three basic steps: initiation, elongation, and termination. Each of these steps will be considered in turn.
Initiation
Initiation involves recognition of the promoter by RNA polymerase. Bacterial promoters generally contain two important DNA sequences that are involved in regulation of transcription. One of these is found at the -10 position (ten base pairs upstream of the transcription start site) and has the sequence TATAAT; the other is found at -35 and has the sequence TTGACA.
RNA polymerase binds to DNA, and scans along the DNA until it encounters a promoter. The sigma subunit recognizes the -35 sequence, and causes the polymerase to bind more tightly. The sigma subunit separates from the polymerase complex, leaving a four subunit core enzyme. The DNA unwinds at the -10 sequence, because that sequence is A/T-rich, and A-T base pairs are weaker than G-C base pairs. The unwound region is known as a transcription bubble, and averages about 18 bp in length. RNA polymerase now begins synthesizing RNA at the appropriate position on the gene.
Elongation
Elongation is much like DNA replication: nucleotides are attached to the 3' end of the growing RNA chain, by the formation of a phosphodiester bond between the 5' phosphate of the incoming nucleotide and the 3' hydroxyl at the end of the RNA chain. There is one major difference between transcription and replication, however. In DNA replication, both DNA strands serve as templates for the synthesis of new DNA. In contrast, only one of the two DNA strands serves as a template for transcription. For a particular gene, it will always be the same strand that serves as a template. This strand is known as the template strand. The other strand, which never serves as a template, is called the non-template or partner strand. As synthesis continues, a transient RNA-DNA hybrid duplex is formed, but the RNA quickly dissociates from the DNA, so that only a few RNA-DNA base pairs are present at a given time. As the polymerase proceeds along the gene, the DNA rewinds itself behind the enzyme. As a result, the transcription bubble appears to move along the gene with the polymerase.
Termination
Termination occurs when the RNA polymerase encounters a termination signal in the gene. Prokaryotic genes have two kinds of termination: rho-independent termination and rho-dependent termination depending on whether termination requires the action of a terminator protein called rho. Each type of termination has a different termination signal in the gene. For rho-independent termination, there is a G/C-rich stretch of nucleotides, followed by an A/T-rich stretch of nucleotides. When the G/C-rich stretch is transcribed into RNA, the sequence of the nucleotides is such that the RNA molecule forms a short double-stranded region called a hairpin:
The hairpin significantly slows down the RNA polymerase (imagine it acting as an anchor or a parachute off the back of the polymerase), and causing it to pause in the A/T-rich region. The weaker A-T base pairs in the RNA-DNA duplex allow the transcription complex to fall apart, ending transcription.
Rho-dependent termination does not involve a specific sequence, although there is a region of the DNA rich in C. As transcription occurs, a protein called rho attaches to the 5' end of the growing RNA molecule, and begins moving along the RNA toward the polymerase. When the polymerase reaches the C-rich sequence, it pauses, and the rho protein is able to catch up to the polymerase and knock it and the newly synthesized RNA off the gene.