Control Mechanisms In Temperate Bacteriophage

Child Health And Human Development

Investigators

Linked publications & trials

Abstract

HK022 uses a novel, RNA-based antitermination mechanism to express many essential genes. A nascent transcript of a viral sequence called put binds to the Elongation Complex (EC) that catalyzed its synthesis and remains associated with it as the transcript elongates. Association with put RNA modifies the EC so that it resists termination at downstream terminators. No other protein factor is required, other ECs in the same cell are unaffected, and there is no apparent terminator specificity. This elongation control pathway appears simpler than others, and its simplicity makes it an attractive target for deeper analysis. How does interaction with put RNA change the properties of the EC?[unreadable] [unreadable] Modification of the EC by put RNA increases the elongation rate and suppresses pausing at a uracil-rich sequence located immediately downstream of the put site. Since pausing is thought to precede termination, and some terminators contain uracil tracts, we decided to investigate the antipausing activity of put at this site. We have shown that the uracil-rich sequence promotes backtracking of the EC. Backtracking is a retrograde movement during which the most recently incorporated nucleotides (at the 3-prime end of the transcript) are melted from the template DNA strand and extruded from RNA polymerase, while compensating amounts of upstream RNA and DNA reenter the EC. We found that put RNA accelerates the EC through the uracil-rich pause by limiting the extent of backtracking. [unreadable] [unreadable] We considered three models of the relationship between the antitermination and antipausing activities of put. (1) Put RNA binds to the EC and then acts in the same way to suppress both pausing and termination. (2) Binding of put RNA to the EC is needed for suppression of termination but not of pausing. Instead, the bulky secondary structure of put RNA suppresses backtracking at the uracil-rich site by sterically inhibiting reentry of nascent RNA into the RNA exit channel of an EC. (3) Binding of put RNA to the EC is required for both antipausing and antitermination, but binding acts in different ways in the two pathways. To suppress pausing, binding anchors the nascent transcript to the EC so that it is unable to reenter the RNA exit channel (see below). To suppress termination, binding confers a persistent modification on the EC. The evidence presented next is consistent with model (3) but not with models (1) and (2).[unreadable] [unreadable] First, we showed that put efficiently suppressed terminators that are located as far as 10 kbp from the put site. By contrast, pausing at the uracil-rich sequence was no longer suppressed when we increased the put-pause distance by as few as 4 bp. Thus, the antipausing activity is local, but the antitermination activity is not. This difference argues strongly that the mechanism of antitermination differs from that of suppression of the uracil-rich pause, contrary to model (1). Second, put RNA folds into two stem-loops that are potentially capable of preventing backtracking by creating a local obstacle. However, studies with anti-put oligonucleotides and put mutants that should not perturb secondary structure argue strongly that formation of a folded structured is not by itself sufficient to prevent backtracking, contrary to model (2). In addition, an RNA polymerase mutant that is unable to bind put RNA is unable to suppress pausing at the uracil-rich sequence. We conclude that put suppression of backtracking at the uracil-rich pause site is the result of a local restrictive effect on retrograde RNA movement that is imposed by EC-bound put RNA. The restriction is relieved as the length of the transcript increases through further elongation. Put remains bound as the EC moves, and this presumably assures the persistence of antitermination. Backtracking of the EC is favored by thermodynamically weak RNA-DNA base pairs, such as that of the ribouridine-deoxyadenosine pair, and such weak hybrid is found at the termination points of intrinsic terminators. Our finding that put does not suppress pausing at distant uracil-rich sequences thus argues that put does not suppress termination by strengthening weak hybrid. [unreadable] [unreadable] The sharply localized nature of the antipausing activity of put allows us to estimate the distance between the end of the RNA exit channel of the EC and position to which put RNA binds. The uracil-rich pause site is located 21 nucleotides from the end of put RNA. Our evidence suggests that at this point, a put-modified EC can backtrack 2 nucleotides. (An unmodified EC can backtrack about 8 nucleotides.) It has been shown by others that about 14 nucleotides of newly synthesized RNA are retained within the EC, partly as RNA-DNA hybrid and partly as a single-stranded chain within the exit channel. These considerations suggest that there are 5 nucleotides (= 21 - 14 - 2) between the outside end of the RNA exit channel and the downstream end of bound put RNA at the point at which backtracking is completely prevented. If we assume that these 5 nucleotides are fully extended and lie along the surface of the EC, the end of bound put RNA is about 3 nm (= 5 nt x 0.6 nm/nt) from the end of the exit channel. The residues of RNA polymerase that are located at this distance include several that belong to a zinc-binding domain that is part of the beta-prime subunit. Mutational and RNA footprinting studies from our laboratory had previously identified this domain as a likely site of put RNA binding.