Using macromolecular crowding agents to identify weak interactions within DNA replication complexes.
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Bacteriophage T4↗
Biomedical subjects
S S Daube
Publications and source records attributed to S S Daube.
Coupling of RNA displacement and intrinsic termination in transcription from synthetic RNA.DNA bubble duplex constructs.
Functional transcription elongation complexes can be formed by adding RNA polymerase in trans to a preformed nucleic acid construct. This construct consists of a double-stranded DNA fragment that contains a noncomplementary (permanent DNA bubble) region into which an RNA primer oligonucleotide has been hybridized. By ligating a DNA fragment containing the strong intrinsic terminator T7Te to the RNA.DNA bubble duplex, we show here that Escherichia coli core RNA polymerase-catalyzed transcription, initiated from such a construct, terminates at the predicted position. Furthermore, we show that the termination efficiency obtained is comparable to that observed in a control reaction initiated with the E. coli holopolymerase from the T7A1 promoter if an RNA oligomer trap is used to permit proper displacement of the nascent RNA from the DNA template strand. The trap oligomer is complementary to the template strand of the permanent DNA bubble and prevents rehybridization of the nascent RNA at this site. Varying the amount of RNA trap that is added permits us to modulate the extent of total RNA displacement. Our results show that RNA displacement and termination efficiency are directly correlated, suggesting that intrinsic termination requires that the nascent RNA be free to assume its solution conformation. Several models of intrinsic termination are presented and discussed in light of these data.
Bacteriophage T7↗
RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes.
Previously [Daube, S.S., & von Hippel, P.H. (1992) Science 258, 1320] we have shown that functional transcription elongation complexes can be formed by adding ribonucleotide triphosphates, Mg2+, and either Escherichia coli or T7 RNA polymerase to synthetic RNA-DNA bubble-duplex constructs. Here these observations are extended to show that the RNA transcripts synthesized from these bubble-duplex constructs are properly displaced from the DNA template during transcription. Some details of the displacement process differ between the polymerases tested. Thus the transcript is fully and processively displaced in the course of T7 polymerase-catalyzed synthesis from the bubble-duplex constructs, while the presence of a large excess of an RNA (or DNA) oligomer complementary to the DNA template sequence within the "permanent" DNA bubble is required to attain complete displacement of the nascent RNA from the construct during synthesis with the core E. coli enzyme. In addition, a correlation is shown between proper RNA displacement and the achievement of full-length transcript synthesis. We conclude that both the T7 polymerase and the E. coli core enzyme actively displace the nascent transcript during elongation and that the requirement for an RNA trap with the E. coli enzyme reflects its slower rate of synthesis. This suggests that these experiments may provide insight into the relative rates of transcript elongation and secondary structure formation within the nascent RNA in elongation and termination. By use of the RNA oligomer trap methodology, multiple rounds of transcript synthesis should be achievable on these bubble-duplex constructs with any polymerase.
Bacteriophage T7↗
Functional transcription elongation complexes from synthetic RNA-DNA bubble duplexes.
A synthetic RNA-DNA bubble duplex construct intended to mimic the nucleic acid framework of a functional transcription elongation complex was designed and assembled. The construct consisted of a double-stranded DNA duplex of variable length (the template and nontemplate strands) containing an internal noncomplementary DNA "bubble" sequence. The 3' end of an RNA oligonucleotide that is partially complementary to the template DNA strand was hybridized within the DNA bubble to form an RNA-DNA duplex with a non-complementary 5'-terminal RNA tail. The addition of either Escherichia coli or T7 RNA polymerase to this construct formed a complex that synthesized RNA with good efficiency from the hybridized RNA primer in a template-directed and processive manner, and displayed other features of a normal promoter-initiated transcription elongation complex. Other such constructs can be designed to examine many of the functional and regulatory properties of transcription systems.
Base Sequence↗
"Macromolecular crowding": thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex.
In vitro biochemical assays are typically performed using very dilute solutions of macromolecular components. On the other hand, total intracellular concentrations of macromolecular solutes are very high, resulting in an in vivo environment that is significantly "volume-occupied." In vitro studies with the DNA replication proteins of bacteriophage T4 have revealed anomalously weak binding of T4 gene 45 protein to the rest of the replication complex. We have used inert macromolecular solutes to mimic typical intracellular solution conditions of high volume occupancy to investigate the effects of "macromolecular crowding" on the binding equilibria involved in the assembly of the T4 polymerase accessory proteins complex. The same approach was also used to study the assembly of this complex with T4 DNA polymerase (gene 43 protein) and T4 single-stranded DNA binding protein (gene 32 protein) to form the five protein "holoenzyme". We find that the apparent association constant (Ka) of gene 45 for gene 44/62 proteins in forming both the accessory protein complex and the holoenzyme increases markedly (from approximately 7 x 10(6) to approximately 3.5 x 10(8) M-1) as a consequence of adding polymers such as polyethylene glycol and dextran. Although the processivity of the polymerase alone is not directly effected by the addition of such polymers to the solution, macromolecular crowding does significantly stabilize the holoenzyme and thus indirectly increases the observed processivity of the holoenzyme complex. The use of macromolecular crowding to increase the stability of multienzyme complexes in general is discussed, as is the relevance of these results to DNA replication in vivo.
Adenosine Triphosphatases↗