Robert Weisberg, PhD, Head, Section on Microbial Genetics
Rodney King, PhD, Guest Worker
Natalia Komissarova, PhD, Staff Fellow
Anu Maharjan, BA, Summer Student
Erik Read, PhD, Postdoctoral Fellow
Tatyana Velikodvorskaya, PhD, Postdoctoral Fellow
Sieghild Sloan, MS, Microbiologist

Temperate bacteriophages can establish a long-term and mutually beneficial association with their bacterial hosts called lysogeny. Lysogeny is a major mechanism of horizontal gene transfer between bacterial families, and temperate bacteriophages constitute a reservoir of genes, such as those encoding virulence factors, that allow their hosts to survive environmental changes. To establish and maintain lysogeny, an infecting phage must control the expression of its own genes so that host viability is unimpaired. Transcription, the first step in gene expression, is catalyzed by DNA-dependent RNA polymerase, an enzyme conserved in all kingdoms of life. After initiating transcription and leaving the promoter, polymerase continues to elongate the transcript until reaching a terminator, where the enzyme, template, and transcript (the elongation complex) dissociate. The efficiency of termination and hence the expression of downstream genes can be controlled by antiterminators. Terminators help silence virus gene expression in lysogens, and antiterminators allow expression of these genes during normal virus growth. The molecular mechanisms of both processes are poorly understood. We study an antitermination mechanism found in bacteriophage HK022, a virus that parasitizes Escherichia coli.

Stable association of put RNA with the elongation complex

King, Sloan, Velikodvorskaya

Antiterminators modify the elongation complex so that it no longer responds to terminators. Together, antiterminators and terminators control gene expression. We study antiterminators that are unusual in that they are embedded in the nascent transcript. These antiterminators, which are encoded by the put sites of E. coli bacteriophage HK022, bind to and modify the elongation complexes that catalyzed their synthesis, thereby suppressing transcription termination and increasing gene expression. We have recently shown that the chemical stability of put RNA is considerably greater than that of the typical E. coli message because the elongation complex protects this RNA from degradation. RNA stability decreased more than 50-fold when mutation prevented binding to the elongation complex. The functional modification conferred by put RNA on the elongation complex is also long-lived: the efficiency of terminator suppression remained high for up to 10 kbp from the putL site. In addition, a cellular ribonuclease rapidly and efficiently cleaved the transcript just downstream of the put sequences, but such cleavage changed neither the stability of put RNA nor the efficiency of antitermination. Thus, the continuity of the RNA that connects put sequences to the growing point (the tether) is not required for persistence of the antiterminating modification in vivo.

These results led us to reinvestigate earlier findings that cleavage of the tether in vitro destabilizes binding of put RNA to the elongation complex. We now find that at least a fraction of put RNA remains bound to the elongation complex in a stable, salt-resistant complex after the tether is cleaved. We are currently studying the antitermination properties of these stable complexes and plan to characterize the binding surfaces.

Suppression of transcriptional pausing by an antiterminator

Komissarova, Sen, Velikodvorskaya

Modification of the elongation complex by binding nascent put RNA increases the elongation rate and suppresses pausing at a uracil-rich sequence located immediately downstream of the put site. We have shown that the uracil-rich sequence promotes "backtracking" of the paused elongation complex. Backtracking is a retrograde movement during which nucleotides at the 3′ end of the transcript are melted from the template DNA strand and extruded from RNA polymerase while compensating amounts of upstream RNA and DNA re-enter the elongation complex. The length of the RNA:DNA hybrid and size of the transcription bubble are maintained during backtracking. Given that the 3′ end of the transcript is distant from the active center of a backtracked elongation complex, elongation cannot resume unless either the complex slides forward to re-engage the 3′ end with the active center or the extruded RNA is cleaved so as to form a new, appropriately positioned 3′ end. We found that put RNA suppresses the uracil-rich pause by suppressing backtracking. However, pausing was no longer suppressed when we increased the distance between the put site and the uracil-rich sequence. In contrast, the suppression of termination by put was not strongly dependent on distance. In addition, the uracil-rich pause site is not required for antitermination. We propose 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 a direct effect of binding of put RNA to the elongation complex. The restriction is alleviated as the elongation complex moves away from the put site (see Figure 16.1).

FIGURE 16.1
Model for suppression of pausing by put.

RNA polymerase (oval) initiates transcription at the promoter and proceeds rightward. The nascent put transcript (gray line), which is synthesized immediately after initiation of transcription, forms two adjacent stem-loops and binds to the elongation complex. When the complex reaches the proximal uracil-rich pause, which is located immediately downstream of the put site in the top template, it is unable to backtrack because of steric constraints. These constraints are removed when the pause is moved to a more promoter-distal location (bottom template). The model accounts for efficient suppression of the proximal but not distal pause by put RNA.

Analysis of a bacteriophage that parasitizes a commensal anaerobe

Read

We have completely sequenced and are now annotating the genome of the bacteriophage B40-8, whose host is Bacteroides fragilis, a human commensal anaerobic bacterium that is frequently pathogenic. The sequence shows that the phage is a very distant relative of all known bacterial viruses. Indeed, about three-quarters of the open reading frames have no known homologues in the sequence database. We plan to determine the transcription pattern of B40-8, with the aim of identifying regulatory factors and sites. In view of the dissimilarity of B40-8 to known organisms, we expect that it will reveal hitherto unknown mechanisms of transcriptional control. We will also determine the function of some of the unknown open reading frames and develop B40-8 as a transducing phage for B. fragilis.

COLLABORATORS

Rodney King, PhD, Western Kentucky University, Bowling Green, KY
Ian Molyneux, PhD, University of Texas, Austin, TX
Ranjan Sen, PhD, Center for DNA Fingerprinting and Diagnostics, Hyderabad, India

For further information, contact weisberr@mail.nih.gov.