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Robert A Weisberg

Publications and source records attributed to Robert A Weisberg.

8 recordsLinked to original sources
PubMed2006

Role of secondary attachment sites in changing the specificity of site-specific recombination.

We previously proposed that lambdoid phages change their insertion specificity by adapting their integrases to sequences found in secondary attachment sites. To test this model, we quantified recombination between partners that carried sequences from secondary attachment sites catalyzed by wild-type and by mutant integrases with altered specificities. The results are consistent with the model, and indicate differential core site usage in excision and integration.

Attachment Sites, Microbiological↗
PubMed2004

A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex.

An evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of DNA-dependent RNA polymerases from bacteria, archaea, and eukaryotes. In bacterial RNA polymerase, this motif, the zinc binding domain, has been implicated in protein-DNA interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. Here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product RNA located upstream of the catalytic center and the RNA-DNA hybrid, a view consistent with structural studies of the elongation complex. We engineered mutations that alter or remove the zinc binding domain of Escherichia coli RNA polymerase. Several mutants, including one that lacked all four zinc ligands and another that lacked the entire domain, produced enzymes that were active in vitro and formed stable elongation complexes. However, they were defective in two functions that require interaction of polymerase with product RNA. First, they terminated less efficiently than the wild-type at intrinsic transcription terminators. Second, enzymes lacking the tip of the zinc binding domain or the zinc ligands did not antiterminate in response to an intrinsic antiterminator encoded by the put site of phage HK022. Termination, but not antitermination, was restored by the bacterial termination factor NusA. Surprisingly, a mutant that lacks the entire zinc binding domain regained a partial response to put. To account for this we suggest that put RNA interacts with an additional site in the elongation complex to mediate antitermination, and that this site is occluded by the wild-type zinc binding domain.

Amino Acid Sequence↗
PubMed2004

Little lambda, who made thee?

The study of the bacteriophage lambda has been critical to the discipline of molecular biology. It was the source of key discoveries in the mechanisms of, among other processes, gene regulation, recombination, and transcription initiation and termination. We trace here the events surrounding these findings and draw on the recollections of the participants. We show how a particular atmosphere of interactions among creative scientists yielded spectacular insights into how living things work.

Bacteriophage lambda↗
PubMed2003

Analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity.

When phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.

Attachment Sites, Microbiological↗
PubMed2003

Suppression of factor-dependent transcription termination by antiterminator RNA.

Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put. put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator.

Bacteriophage HK022↗
PubMed2002

Take your vitamins with a pinch of RNA.

RNA "aptamers" capable of binding and discriminating among structurally related small molecules can be concocted in the laboratory. Two groups have now discovered that conserved domains in the 5' ends of some mRNAs bind specific metabolites and respond by changing their shape in biologically useful ways, demonstrating that aptamers also are present in the natural world.

5' Untranslated Regions↗
PubMed2002

Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase.

The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination. To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability. All the mutants were defective in put-mediated antitermination. The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA. Some, but not all, mutants distinguished between put variants that differed in this region. This suggests that the Zn-finger motif interacts directly and specifically with put RNA. All the mutants in the basic residues complemented a temperature-sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.

Base Sequence↗