Bacterial RNA Polymerase Terminates at Discrete Sites

KEY CONCEPTS
- Two classes of terminators have been identified: Those recognized solely by RNA polymerase itself without the requirement for any cellular factors are usually referred to as intrinsic terminators. Others require a cellular protein called rho and are referred to as rho-dependent terminators.
- Intrinsic termination requires recognition of a terminator sequence in DNA that encodes a hairpin structure in the RNA product.
- The signals for termination lie mostly within sequences already transcribed by RNA polymerase, and thus termination relies on scrutiny of the template and/or the RNA product that the polymerase is transcribing.
Once RNA polymerase has started transcription, the enzyme moves along the template, synthesizing RNA. As described earlier in this chapter in the section titled The Transcription Reaction Has Three Stages, movement is not at a steady pace; the rate varies and is determined by the sequence context. The RNA polymerase can pause or arrest and even backtrack, either of which can lead to termination. The enzyme stops adding nucleotides to the growing RNA chain, releases the completed product, and dissociates from the DNA template at the point of a genuine terminator sequence or during a prolonged pause. Termination requires that all hydrogen bonds holding the RNA–DNA hybrid together must be broken, after which the DNA duplex reforms.
It is sometimes difficult to define the termination site for an RNA that has been synthesized in the living cell, because the 3′ end of the molecule can be degraded by a 3′ exonuclease or cleaved by an endonuclease, leaving no history of the actual site at which RNA polymerase terminated in the remaining transcript; in fact, specific 3′-end modifications are part of normal RNA processing in eukaryotes. Therefore, termination sites are often best characterized in vitro. The ability of the enzyme to terminate in vitro, however, is strongly influenced by parameters such as the ionic strength and temperature at which the reaction is performed; as a result, termination at a particular position in vitro does not prove that this is the same site where it occurs in cells. If the same 3′ end is detected in vivo and with purified components in vitro, though, this is generally recognized as good evidence for the authentic site of termination.
FIGURES 1and 2 summarize the two major features found in intrinsic terminators. First, intrinsic terminators—that is, those that do not require auxiliary rho factor (ρ), as described shortly—require a G+C–rich hairpin to form in the secondary structure of the RNA being transcribed. Thus, termination depends on the RNA product and is not determined simply by scrutiny of the DNA sequence during transcription. The second feature is a series of up to seven uracil residues (thymine residues in the DNA) following the hairpin stem but preceding the actual position of termination. Approximately 1,100 sequences in the E. coli genome fit these criteria, suggesting that more than half of the cell’s transcripts are terminated at intrinsic terminators. Rho-dependent terminators are defined by the need for addition of rho factor in vitro, and mutations show that the factor is involved in termination in vivo.

FIGURE 1. The DNA sequences required for termination are located upstream of the terminator sequence. Formation of a hairpin in the RNA may be necessary.

FIGURE 2. Intrinsic terminators include palindromic regions that form hairpins varying in length from 7 to 20 bp. The stem-loop structure includes a G-C–rich region and is followed by a run of U residues.
Terminators vary widely in their efficiencies. Readthrough transcripts refer to the fraction of transcripts that are not stopped by the terminator. (Readthrough is the same term used in translation to describe a ribosome’s suppression of termination codons.) Furthermore, the termination event can be prevented by specific ancillary factors that interact with RNA and/or RNA polymerase, a situation referred to as antitermination. Thus, as in the case of initiation or elongation, termination can be regulated as a mechanism for controlling gene expression.
Initiation and termination also have other parallels. Both require breaking of hydrogen bonds (initial melting of DNA at initiation and RNA–DNA dissociation at termination), and both can utilize additional proteins (sigma factors, activators, repressors, and rho factor) that interact with the core enzyme. Whereas initiation relies solely upon the interaction between RNA polymerase and duplex DNA, the termination event also involves recognition of signals in the transcript by RNA polymerase.
Point mutations that reduce termination efficiency usually occur within the stem region of the hairpin, replacing GC base pairs with weaker AT base pairs, or in the U-rich sequence, supporting the importance of these sequences in the mechanism of termination.
The RNA–DNA hybrid makes a large contribution to the forces holding the elongation complex together. Thus, breaking the hybrid would destabilize the elongation complex, leading to termination. Interactions of the hairpin with the RNA polymerase or forces exerted by formation of the hairpin as the RNA emerges from the RNA exit channel can transiently misalign the 3′ end of the RNA with the active center in the enzyme. This misalignment, combined with the unusually weak RNA–DNA hybrid formed from the rU-dA RNA–DNA base pairs resulting from the stretch of U residues, destabilize the elongation complex.
Termination efficiency in vitro can vary widely, though, from 2% to 90%. The efficiency of termination depends not only on the sequences in the hairpin and the number and positions of U residues downstream of the hairpin but also on sequences both further upstream and downstream of the site of termination.
Instead of terminating, the enzyme may simply pause before resuming elongation. These pause sites can serve regulatory purposes on their own . Whether RNA polymerase arrests and releases the RNA chain or whether it merely pauses before resuming transcription (i.e., the duration of the pause and the efficiency of escape from the pause) is determined by a complex set of kinetic and thermodynamic considerations resulting from the characteristics of the hairpin and the U-rich stretch in the RNA and the upstream and downstream sequences in the DNA. For example, pausing can occur at sites that resemble terminators, but where the separation between the hairpin and the U-run is longer than optimal for termination.