MECHANISMS FOR CONTROLLING GENE EXPRESSION IN BACTERIA AND BACTERIOPHAGE
, Head, Section on Microbial Genetics
Natalia Komissarova, PhD, Staff Scientist
Erik Read, PhD, Postdoctoral Fellow
Tatyana Velikodvorskaya, PhD, Visiting Fellow
Sieghild Sloan, MS, Microbiologist
Jisha Chaliserry, MS, Graduate Student
Temperate bacteriophages can establish lysogeny, a long-term and mutually beneficial association with their bacterial hosts. Lysogeny is a major mechanism of horizontal gene transfer between bacterial families, and temperate bacteriophages are reservoirs of genes that allow their hosts to survive environmental challenges. To establish and maintain lysogeny, a temperate phage controls the expression of its own genes so that host viability is unimpaired. Many phages maintain host viability by regulating the elongation step of transcription. Transcription is catalyzed by DNA-dependent RNA polymerase (RNAP). After initiating polymerization on a DNA template, the transcript is elongated until the polymerases reaches a terminator, where the enzyme, template, and transcript (the elongation complex; or EC) dissociate from each other. The efficiency of termination and, hence, the expression of downstream genes can be controlled by antiterminators. The molecular mechanisms of termination and antitermination are poorly understood. We aim to understand these mechanisms by studying transcription directed by an Escherichia coli phage, HK022, that uses a robust yet relatively simple antitermination pathway to control gene expression. We have recently expanded our work by studying transcriptional regulatory mechanisms in a different temperate phage, B40-8, that parasitizes Bacteroides fragilis, a bacterium distantly related to E. coli.
Suppression of transcriptional pausing by an antiterminator
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 EC that catalyzed its synthesis and remains associated with it as the transcript continues to elongate. Association with put RNA modifies the EC so that it elongates faster and resists termination at downstream terminators. No other protein factor is absolutely required, other ECs in the same cell are unaffected, and there is no obvious terminator specificity. The apparent simplicity of this antitermination pathway makes it an attractive target for deeper analysis.
Modification of the EC by put RNA suppresses pausing at a U-rich sequence located close to the put site. Given that pausing is thought to precede termination and that intrinsic terminators contain U tracts at the termination point, we decided to investigate put-mediated antipausing at this site. We showed that the U-rich sequence promotes “backtracking” of the EC. Backtracking is a retrograde movement during which the nucleotides at the 3¢ end of the transcript are melted from the template DNA strand and extruded from RNAP while compensating amounts of upstream DNA and nascent RNA re-enter the EC. Backtracking is favored by the formation of thermodynamically weak rU-dA base pairs (bp). We found that put RNA accelerates the EC through the U-rich pause by limiting the extent of backtracking.
Figure 7.4
Models of the relation between antipausing and antitermination. The grey oval represents the surface of RNAP, and the two stem-loops respresent folded put RNA. Arrowheads indicate the promoter and the RNA growing point (3′ end). The DNA template is represented by two parallel lines that diverge in the region of the transcription bubble, where nascent RNA is hybridized to the template strand of DNA. The hatching in models 1 and 3 indicates modification of the EC.
We considered three models of the relation between antitermination and antipausing (Figure 7.4). (1) Put RNA binds to the EC and then acts in the same way to suppress pausing and termination. (2) Binding is required for the suppression of termination but not for suppression of pausing. Instead, the bulky secondary structure of put RNA suppresses backtracking at the U-rich site by sterically blocking re-entry of nascent RNA into the EC. (3) Binding is required for both antipausing and antitermination, but the bound RNA acts in different ways in the two pathways. Pausing is suppressed because the bound RNA is unable to re-enter the EC while termination is suppressed through a stable put-mediated structural modification of the EC. Our evidence is consistent with model 3 but not with models 1 and 2.
First, we showed that put efficiently suppressed distant terminators but no longer suppressed the U-rich pause when the put-pause distance was increased by only 4 bp. Thus, antipausing is local, but antitermination is not, contrary to model 1. Second, studies with anti-put oligonucleotides, put mutants that should not perturb secondary structure, and an RNAP mutant that cannot bind to put RNA showed that RNA secondary structure is not sufficient to prevent backtracking, contrary to model 2. We conclude that put suppression of backtracking is the result of a local restrictive effect on retrograde RNA movement 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, thus ensuring the persistence of antitermination. Our finding that put does not suppress pausing at distant U-rich sequences argues that put does not suppress termination by strengthening the weak rU-dA hybrid.
Newly synthesized RNA leaves the EC through an exit channel. The sharply localized nature of the antipausing activity of put and the assumptions of model 3 allow an estimate of the approximate distance between the end of the RNA exit channel and the put RNA binding site. We suggest that there are five nucleotides between these points when the put-modified EC reaches the position at which no backtracking can occur [heavy line in Figure 7.4, model 3]. Five nucleotides should have a contour length of about 3 nm when fully extended on the surface of the EC. The residues of RNAP that are located at this distance include several that belong to a zinc-binding domain that we had previously identified as a likely site of put RNA binding on the basis of genetic studies.
Analysis of a bacteriophage parasitizing a commensal anaerobe
We have sequenced and annotated the genome of bacteriophage B40-8, whose host is Bacteroides fragilis, a human commensal anaerobe that is frequently pathogenic. The sequence reveals a circular genome of 45,805 bp that encodes 66 genes, all in the same orientation (Figure 7.5). Homology searches show that the virus is a distant relative of known bacterial viruses, as only 16 open reading frames have significant similarity to sequences within the NCBI database. The similarities suggest that some genes encode functions known to be important for bacteriophage replication, morphogenesis, and lysis. Genes 1 and 2 appear to encode a terminase used for packaging linear concatameric chromosomal DNA into phage heads by a headful mechanism. Phylogenetic comparisons show that the putative terminases are distantly related to known terminases, suggesting that the mechanism of DNA cutting is distinct. Indeed, we have identified the chromosomal location of the left cut to be precisely at bp 12,013, whereas the right cut occurs over a 250 bp region within gene 22. The precision of the first cut is not seen in other bacteriophages, but the variability of the second cut shows that the headful mechanism of packaging may be exploited to develop the phage as a tool for generalized transduction. The distribution of closely associated transcription terminators and promoters reveals a new strategy for gene regulation not seen in known bacterial viruses. Further analysis during the infection cycle will reveal gene expression patterns and regulatory circuits, thus permitting a deeper understanding of transcriptional control in this virus and its host.
Figure 7.5
A linear map of the B40-8 genome highlighting predicted genes:
, rho-independent transcription terminators:
, rho-independent terminators fused to promoters:
, and the packaged chromosomal termini:
.
Rutkai E, György A., Dorgai L, Weisberg RA. Role of secondary attachment sites in changing the specificity of site-specific recombination. J Bacteriol 2006;188:3409-11.
Sloan S, Rutkai E, King RA, Velikodvorskaya T, and Weisberg RA. Protection of antiterminator RNA by the transcript elongation complex. Mol Microbiol 2007;63:1197-208.
Weisberg RA, Hinton DM, Adhya S. Regulatory strategies in bacteriophage growth and lysogeny. In: Branford D, series ed. The Encyclopedia of Virology, Elsevier, in press.
COLLABORATORS
Rodney King, PhD, Western Kentucky University, Bowling Green, KY
Ian Molineux, PhD, University of Texas, Austin, TX
Ranjan Sen, PhD, Center for DNA Fingerprinting and Diagnostics, Hyderabad, India
For further information, contact rweisberg@nih.gov.
