Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

Abstract

RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among ␥-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.RNase P ͉ tmRNA ͉ long-range helix B ecause of the degeneracy and stability of base-pairing, RNA has a high propensity to form long-lived undesirable intermediates along their folding pathways (1-3). Particularly challenging for RNA folding during transcription is the formation of duplexes composed of two strands located far apart in sequence. In the time before the transcription of the downstream strand, the upstream strand can form stable yet nonnative base pairs with other regions, which could hinder folding. This undesirable possibility can be circumvented by sequestering the upstream strand in specific but labile structures. Such structures can guide the formation of the native long-range helices by avoiding the formation of long-lived unproductive species.When compared with Mg 2ϩ -induced refolding of full-length RNAs, two obvious properties that influence RNA folding during transcription are the 5Ј to 3Ј polarity of RNA synthesis and the transcriptional speed of the RNA polymerase (4-8). In addition, bacterial RNA polymerases pause during transcriptional elongation (9, 10). The location and/or duration of the pause sites can alter folding (7, 11). A previous work on the folding during transcription of a circularly permuted version of the Bacillus subtilis RNase P RNA demonstrated that pausing at a specific site alters its folding during transcription by Escherichia coli RNA polymerase (11). Only the pause at this site was required for the accelerated folding of one of the two domains of P RNA; varying the transcriptional speed had no effect. Pausing at this site altered the nascent RNA so that an undesirable structure involving a region downstream to the pause site was avoided.Although the above result demonstrated that pausing can influence folding, it did not address the question of biological relevancy, because (i) circularly permuted RNA transcripts are unnatural, having changed t...

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…This polarity-driven folding process is further controlled through the strategic location of transcriptional pause sites (46) that presumably allow sufficient time for secondary and tertiary interactions to take place. These interactions are often important for the folding of complex RNA structures (47,48). Transcriptional pausing has been identified in metabolite-binding riboswitches responsive to flavin mononucleotide (49), adenine (27), and AdoCbl (19) and in a pH-responsive riboregulator (50).…”

Folding Kinetics of Large RNAs

“…In a cellular context, furthermore, the nascent RNA molecule starts to fold before the transcription process is completed. Co-transcriptional folding is strongly affected by the speed of elongation, sitespecific pausing of the RNA polymerase and interactions of the nascent RNA molecule with proteins or small-molecule metabolites (1), (2). Since transcription is a sequential process, the 5' region of a native helix is synthesized before its 3' portion.…”

Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

“…RNAP pauses at strategic sites located downstream of the 5′ region of all long-range helices. These pauses enable nascent RNAs to fold into beneficial nonnative structures wherein the 5′ region is sequestered and poised for subsequent folding of the native structure (13). Second, riboswitch function involves two or more competing structures; one structure directly senses cellular metabolite concentration.…”