RNA polymerase is a highly processive macromolecular machine. Structural factors that help to maintain the transcription elongation complex as an intact unit when it is paused include: the sliding clamp that binds the DNA template in front of the core RNA polymerase; The RNA exit tunnel that holds the nascent RNA and the segment of nascent RNA that is held to the DNA template by hydrogen bonds. Specific mechanisms are required to release RNA polymerase from the TEC. Two transcription termination pathways, intrinsic termination and Rho-dependent termination, contribute about equally to this release in E. coli.
The intrinsic termination pathway is so named because it takes advantage of the core RNA polymerase's intrinsic catalytic activity to terminate transcription. Although nucleotide sequences specifying intrinsic terminators are present on DNA, it is actually the nascent, RNA (and not DNA) that triggers the transcription termination response. Two sequence motifs on the nascent RNA strand are essential for intrinsic terminator function.
The first motif, a G-C rich inverted repeat, allow the RNA to fold into a stem and loop structure that reaches to within seven to nine nucleotides of the 3'-end of the nascent RNA strand. Mutations that maintain the stable stem structure are usually tolerated. Those that decrease the stem structure's stability tend to reduce or eliminate termination. Multiple mutations within the stem region also may lead to loss of terminator activity even though the stem structure retains its stability. Therefore, secondary RNA structure is important in determining intrinsic terminator activity but nucleotide sequence within the stem also appears to make a contribution.
The second motif, a run of eight to ten nucleotides that consists mostly of uridines, comes immediately after the stem and loop structure. Intrinsic terminators appear to act by first causing the transcription elongation complex to pause and then to release the nascent RNA chain. Replacing the uridines in the second motif with other nucleotides converts an intrinsic terminator into a pause signal.
The Rho-dependent transcription termination pathway requires a hexameric helicase called the Rho factor. Jeffrey W. Roberts originally detected the Rho factor in purified bacterial extracts because of the factor's ability to terminate transcription of bacteriophage lambda genes. Rho factor, which consists of six identical subunits (subunit molecular mass = 46 kDa), can be detected in vitro as an ATP-dependent helicase that releases RNA from an RNA-DNA hybrid. Each subunit has an N-terminal domain (residues 1-130) that contains the primary binding site for single-stranded RNA and a C-terminal domain (residues 131-419) that contains a secondary binding site for single-stranded RNA. The C-terminal domain also has an ATP binding site that is essential for helicase activity. Under physiological conditions Rho factor loads onto nascent mRNAs at a cytosine-rich region that contains 40 or more nucleotides known as the Rho utilization site.