Direct Detection of Abortive RNA Transcripts in Vivo

Abstract

During transcription initiation in vitro, RNA polymerase can engage in abortive initiation-the synthesis and release of short, 2 to 15 nucleotide, RNA transcripts-prior to productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detect abortive transcripts in vivo and thereby show that abortive initiation occurs in vivo. We further show that abortive initiation in vivo is a determinant of promoter strength, a determinant of RNA polymerase function, and a target of regulation by transcription factor GreA. Abortive transcripts may have functional, physiologically important roles in regulating gene expression in vivo.During transcription, RNA polymerase (RNAP) synthesizes the first ∼8-15 nucleotides (nt) of RNA as an RNAP-promoter initial transcribing complex (1-3) [using a "scrunching" mechanism (4)]. Upon synthesis of an RNA transcript of a threshold length of ∼8-15 nt, RNAP breaks its interactions with promoter DNA, escapes the promoter, and enters into processive synthesis of RNA as an RNAP-DNA transcription elongation complex (1-3) [using a "stepping" mechanism (5)]. In transcription reactions in vitro, the RNAP-promoter initial transcribing complex can engage in tens to hundreds of cycles of synthesis and release of short RNA transcripts ("abortive initiation") (1-3,6-8). Abortive initiation competes with productive initiation in vitro and, as such, is a critical determinant of promoter strength and a target of transcription regulation in vitro (1-3,7-13). It has been proposed that abortive initiation likewise occurs in vivo (6,8). In support of this proposal, factors that affect yields of full-length transcripts in vitro through effects on abortive initiation likewise affect yields of full-length transcripts in vivo (9-14). However, no direct evidence that abortive initiation occurs in vivo has been presented.The bacteriophage T5 N25 promoter and its derivative N25anti are classic model systems for the study of abortive initiation and promoter escape (9,(11)(12)(13)(14). N25 and N25anti differ only in their initial transcribed sequences (positions +3 to +20) but exhibit radically different characteristics in vitro with respect to the abortive:productive ratio (APR; 40 for N25; ∼300 for N25anti), the maximum size of abortive transcripts (10 nt for N25; 15 nt for N25anti), and the kinetics of promoter escape (k clear ∼1.7 per min for N25; ∼0.06 per min for N25anti).To determine whether abortive initiation occurs in vivo, we sought to detect abortive transcripts generated during transcription from a plasmid-borne copy of N25anti in Escherichia coli, using locked nucleic acid probes developed for hybridization-based detection of microRNAs (15,16). We reasoned that the relatively high APR of N25anti would facilitate accumulation of detectable quantities of abortive transcripts and that the relatively high maximum size of

Crystal Structure of the Human Primase

“…9A), and the counting effect almost completely disappeared. The presence of relatively intense bands represented by 2-4-nucleotide primers indicates the high probability of primase dissociation from short primers, which is probably due to the abortive synthesis characteristic of RNA polymerases (45). We conclude that the ability of primase to count the oligo(dT) or oligo(dA) primer length depends primarily on template sequence and to a lesser extent on other factors, including structural features of the primase molecule.…”

New Insights into the Mechanism of Initial Transcription

“…Indeed, short abortive products have been observed both in vitro and in vivo (6), and the transition from initially transcribing to stably elongating complexes is a target of gene regulation in many systems, most notably in promoter proximal pausing (7).…”

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

“…However, the fact that larger amounts of the optimized parA mRNA were observed both in the one-copy and 14-copy parA systems (Fig. 5) may indicate that the new 5′-UTR mRNA structure is probably (Hsu et al 2006;Berg et al 2009;Goldman et al 2009) (i) increasing the stability of the complex RNA polymerase-transcript and thus reducing the amounts of abortive transcripts, (ii) decreasing the susceptibility to degradation of the transcript, and (iii) decreasing the availability to degradation because the transcript binds longer to rRNA. Thus, the final amount of the resolvase in the system is a compromise between transcriptional and translational rates.…”