Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient recognition of DNA is a prerequisite for the development of biological effectors, including transcription and translation regulators, transfection vectors, and DNA sensors. To provide an effective scaffold for multivalent interactions with DNA, we have fabricated mixed monolayer protected gold clusters (MMPCs) functionalized with tetraalkylammonium ligands that can interact with the DNA backbone via charge complementarity. Binding studies indicate that the MMPCs and DNA form a charge-neutralized, nonaggregated assembly. The interactions controlling these assemblies are highly efficient, completely inhibiting transcription by T7 RNA polymerase in vitro.

Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1). Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription. The coding and noncoding strands appear to make different contributions to promoter recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km. In contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat. Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed ‘nontemplated addition,’ also occurs via templated primer extension. At last, this work demonstrates the utility of RNA-Seq as a tool for in vitro mechanistic studies, providing information far beyond that provided by traditional gel electrophoresis.

Light and life: A photolabile gold nanoparticle has been constructed to serve as a DNA carrier. UV irradiation causes the reversal of the nanoparticle surface charge, resulting in effective DNA release and reactivation of suppressed DNA transcription in vitro. This effect was also observed in living cells, together with efficient internalization of DNA into the nucleus.