12. More subunits for RNA polymerase
9.12 More subunits for RNA polymerase |
Termination and antitermination are closely connected, and involve bacterial proteins and phage proteins that interact with RNA polymerase in response to sequences within certain transcription units. A lambda nut site consists of two sequence elements, called boxA and boxB. Sequence elements related to boxA are also found in bacterial operons. boxA is required for binding bacterial proteins that are necessary for antitermination in both phage and bacterial operons. boxB is specific to the phage genome, and mutations in boxB abolish the ability of pN to cause antitermination.
The discovery of antitermination as a phage control mechanism led to the identification of further components of the transcription apparatus. The bacterial proteins with which pN interacts can be identified by isolating mutants of E. coli in which pN is ineffective. Several of these mutations lie in the rpoB gene. This argues that pN (like rho factor) interacts with the β subunit of the core enzyme.
Other E. coli mutations that prevent pN function identify the nus loci: nusA, nusB, nusE, and nusG. (The term "nus" is an acronym for N utilization substance.) The nus loci code for proteins that form part of the transcription apparatus, but that are not isolated with the RNA polymerase enzyme in its usual form. The nusA, nusB, and nusG functions are concerned solely with the termination of transcription. nusE codes for ribosomal protein S10; the relationship between its location in the 30S subunit and its function in termination is not clear. NusA is a general transcription factor that increases the efficiency of termination, probably by enhancing RNA polymerase’s tendency to pause at terminators (and indeed at other regions of secondary structure; see below). NusB and S10 form a dimer that binds specifically to RNA containing a boxA sequence. NusG may be concerned with the general assembly of all the Nus factors into a complex with RNA polymerase. A distinction in the requirements for Nus functions is that NusA suffices for pN to prevent termination at intrinsic terminators, whereas all 4 Nus functions are required for pN action at rho-dependent terminators.
Antitermination occurs in the rrn (rRNA) operons of E. coli, and involves the same nus functions. The leader regions of the rrn operons contain boxA sequences; NusB-S10 dimers recognize these sequences and bind to RNA polymerase as it elongates past boxA. This changes the properties of RNA polymerase in such a way that it can now read through rho-dependent terminators that are present within the transcription unit.
The boxA sequence of lambda RNA does not bind NusB-S10, and is probably enabled to do so by the presence of NusA and pN; the boxB sequence could be required to stabilize the reaction. So variations in boxA sequences may determine which particular set of factors is required for antitermination. The consequences are the same: when RNA polymerase passes the nut site, it is modified by addition of appropriate factors, and fails to terminate when it subsequently encounters the terminator sites.
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Figure 9.34 RNA polymerase may alternate between initiation-competent and termination-competent forms as sigma and Nus factors alternatively replace one another on the core enzyme. |
NusA binds to the polymerase core enzyme, but does not bind to holoenzyme. When sigma factor is added to the α 2ββ′NusA complex, it displaces the NusA protein, thus reconstituting the α 2ββ′σ holoenzyme. This suggests that RNA polymerase passes through the cycle illustrated in Figure 9.34, in which it exists in the alternative forms of an enzyme ready to initiate (α 2ββ′σ) and an enzyme ready to terminate (α 2ββ′σNusA). When the holoenzyme (α 2ββ′σ) binds to a promoter, it releases its sigma factor, and thus generates the core enzyme (α 2ββ′σ) that synthesizes RNA. Then a NusA protein recognizes the core enzyme and binds to it, generating the α 2ββ′σNusA complex. While the α 2ββ′σNusA polymerase is bound to DNA, the Nus components cannot be displaced. But when termination occurs, the enzyme is released in a state in which the Nus factors either are released or can be displaced by sigma factor.
Core enzyme therefore alternates between associating with sigma for initiation and associating with Nus factors for termination. Sigma and the Nus factors are mutually incompatible associates of the core. There seems no reason to regard either one as any more a component than the other; we may regard them as alternative subunits. So the core enzyme represents a minimal form of RNA polymerase, competent to engage in the basic function of RNA synthesis, but lacking subunits necessary for other functions. It is a moot point where RNA polymerase ends and the wider transcription apparatus begins.
Antitermination requires pN to bind to RNA polymerase in a manner that depends on the sequence of the transcription unit. Does pN recognize the boxB site in DNA or in the RNA transcript? It does not bind directly to either type of sequence, but does bind to a transcription complex when core enzyme passes the boxB site. Probably it recognizes the boxB RNA sequence, but also must make protein-protein contacts with NusA and with RNA polymerase in order to bind. After joining the transcription complex, pN remains associated with the core enzyme, in effect becoming an additional subunit whose presence changes recognition of terminators. It is possible that pN in fact continues to bind to both the boxB RNA sequence and to RNA polymerase, maintaining a loop in the RNA; thus the role of boxB RNA would partly be to tether pN in the vicinity, effectively increasing its local concentration.
What is the antiterminating action of pN? It might act to prevent RNA polymerase from pausing, thus denying rho factor the opportunity to cause termination; or it might act directly to antagonize the ability of rho to release the nascent RNA transcript. pN indeed has an antipausing activity; it is not clear yet whether it also acts on rho factor.
pQ, which prevents termination later in phage infection by acting at qut, has a different mode of action. The qut sequence lies at the start of the late transcription unit. The upstream part of qut lies within the promoter, while the downstream part lies at the beginning of the transcribed region. This implies that pQ action involves recognition of DNA, and implies that its mechanism of action, at least concerning the initial binding to the complex, must be different from that of pN.
The basic action of pQ is to interfere with pausing; and once pQ has acted upon RNA polymerase, the enzyme shows much reduced pausing at all sites, including rho-dependent and intrinsic terminators. So pQ does not act directly on termination per se, but instead allows the enzyme to hurry past the terminator, thus depriving the core polymerase and/or accessory factor of the opportunity to cause termination.
The general principle is that RNA polymerase may exist in forms that are competent to undertake particular stages of transcription, and its activities at these stages can be changed only by modifying the appropriate form. So substitutions of sigma factors may change one initiation-competent form into another; and additions of Nus factors may change the properties of termination-competent forms.
Termination seems to be inextricably connected with the mode of elongation. In its basic transcription mode, core polymerase is subject to many pauses during elongation; and pausing at a terminator site is the prerequisite for termination to occur. Under the influence of factors such as NusA, pausing becomes extended, increasing the efficiency of termination; while under the influence of pN or pQ, pausing is abbreviated, decreasing the efficiency of termination. Because recognition sites for these factors are found only in certain transcription units, pausing and consequently termination are altered only in those units.
