Processive Antitermination

After initiating synthesis of RNA at a promoter, RNA polymerase (RNAP) normally continues to elongate the transcript until it reaches a termination site. Important elements of termination sites are transcribed before polymerase translocation stops, and the resulting RNA is an active element of the termination pathway. Nascent transcripts of intrinsic sites can halt transcription without the assistance of additional factors, and those of Rho-dependent sites recruit the Rho termination protein to the elongation complex. In both cases, RNAP, the transcript, and the template dissociate (reviewed in references 76 and 80). Termination is rarely, if ever, completely efficient, and the expression of downstream genes can be controlled by altering the efficiency of terminator readthrough. Two distinct mechanisms of elongation control have been reported for bacterial RNA polymerases. In one, exemplified by attenuation of the his and trp operons of Salmonella typhimurium and Escherichia coli, respectively, a single terminator is inactivated by interaction with an upstream sequence in the transcript, with a terminator-specific protein, or with a translating ribosome that follows closely behind RNAP (reviewed in references 35 and 104). In a second, whose prototype is antitermination of phage l early transcription, polymerase is stably modified to a terminator-resistant form after it leaves the promoter. In this case, the modified enzyme not only transcribes through sequential downstream terminators, but also it is less sensitive to the pause sites that normally delay transcript elongation. Both pathways are widespread in nature, but in this minireview we consider only the second, which is known as processive antitermination (for previous reviews, see references 22, 23, 27, and 32). The recent explosive growth in our understanding of transcription elongation (reviewed in references 57, 96, and 99) make this an especially appropriate time to survey regulatory elements that target the transcription elongation complex. Antitermination in l is induced by two quite distinct mechanisms. The first is the result of interaction between l N protein and its targets in the early phage transcripts, and the second is the result of an interaction between the l Q protein and its target in the late phage promoter. We describe the N mechanism first. Lambda N, a small basic protein of the arginine-rich motif (ARM) (Fig. 1) family of RNA binding proteins, binds to a 15-nucleotide (nt) stem-loop called BOXB (17) (Fig. 2). (We will capitalize the names of sites in RNA and italicize the names of the corresponding DNA sequences; e.g., BOXB and boxB.) boxB is found twice in the l chromosome, once in each of the two early operons (82, 83). It is close to the start point of the PL operon transcript and just downstream of the first translated gene of the PR operon. Neither the distance between the transcription start site and boxB, nor the nature of the promoter (at least in the case of sigma-70-dependent promoters), nor the nature of the terminator is relevant to N action. Although the boxB sequence is not well conserved in other bacteriophages of the l family, most of these phages encode proteins that are analogous to l N and have sequences capable of forming BOXB-like structures in their PL and PR operons. In some cases, it has been shown that these structures are recognized by the cognate N analogs. It is believed that this accounts for the phage specificity of N-mediated antitermination. The structures of complexes between the ARMs of l N (residues 1 to 22) and P22 N (residues 14 to 32) and their cognate BOXBs have recently been determined by nuclear magnetic resonance (15, 48) (Fig. 2). The two complexes, although similar, show differences that account for the specificity of N-BOXB recognition (92, 101). Upon binding, the l and P22 ARMs adopt a bent a-helix conformation that packs against the BOXB hairpin through hydrophobic and ionic interactions. Residues in the amino-proximal segments make multiple base, ribose, and phosphate contacts in the 59-ascending stem of BOXB without disrupting its regular A-form. The two stem-proximal residues of both RNA loops form a sheared G z A base pair which is contacted by R7 of l N and R19 of P22 N (note that R19 corresponds to Arg 6 of Fig. 2; see Fig. 2 legend). In the l complex, the fourth residue (G) of the GAAGA loop is extruded and the remaining residues form a GNRA fold similar to the base-stacked GAAA tetraloop reported in a number of important RNA structures (Fig. 2). P22 N also creates a GNRA fold, but this is accomplished by extrusion of the third residue (C) of the GACAA loop. In contrast to the extruded G residue in BOXB-l, which is not close to l N, the extruded C residue in BOXB-P22 makes contacts with residues in the carboxyl-proximal segment of P22 N. The structure of the l N complex is stabilized by an important stacking interaction between W18 and the second residue (A) in the BOXB-l loop. This interaction is not found in the P22 complex. Formation of the GNRA fold is essential for l N binding, and mutations in loop residues 1, 3, and 5 that prevent tetraloop formation block N binding (17). The structure of the complex between the BOXB of phage 21 and its cognate N protein must be substantially different than the complexes described above because the BOXB-21 loop (TCTAACCG) cannot be folded into a GNRA tetraloop. However, the HK022 Nun protein, which recognizes l BOXB (see below), probably does so in a way that resembles that of l (18). If so, A3, R7, * Corresponding author. Mailing address: Section on Microbial Genetics, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2785. Phone: (301) 496-3555. Fax: (301) 496-0243. E-mail: wia@cu.nih. gov.