Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded

Abstract

RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked. Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3 end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme. The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage. Secondary structure elements or the 5 end of the transcript can prevent the isomerization by blocking the RNA threading. The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.Arrested ternary complexes of prokaryotic and eukaryotic RNA polymerases (RNAPs) retain the RNA but lose catalytic activity (1-4). DNA sites that provoke arrest do not show strong sequence similarity except that they are enriched in homopolymeric oligo(T) tracts that are 7-10 nucleotides long (4-6). However, transcription through certain regions in DNA is particularly predisposed to arrest. At such sites, the front end of the RNAP footprint appears to remain fixed on the template while RNA chain growth continues (5, 7-10). These unusual regions were called sites of discontinuous elongation, because normally RNAP footprints translocate monotonously along the template as each nucleotide is added to the RNA.Being a potential block for RNAP passage through a transcription unit, arrest may affect gene expression by decreasing formation of full-sized transcripts, as documented in vitro for Escherichia coli RNAP transcribing the rrnB gene (3) and for eukaryotic RNA polymerase II (Pol II) transcribing histone H3.3, and several other genes (3, 4, 11). In addition, arrested complex formation represents a side pathway in normal termination, since a fraction of the ternary complexes at prokaryotic and intrinsic Pol II termination sites fall into an arrested state instead of dissociating from the template (4, 5).Elongation factors TFIIS in eukaryotes and GreB in prokaryotes relieve arrest by inducing internal cleavage of the transcript (3,6,(12)(13)(14)(15). The 3Ј-proximal product of the cleavage may reach 10-20 nucleotides in length, whereas in active complexes the analogous products are only 2-3 nucleotides long (3,6,16,17). The cleaved RNA fragments dissociate from the complex, which allows RNAP to resume elongation in the correct DNA-RNA register from the newly formed 3Ј end. No movement of RNAP was detected upon transcript cleavage in arrested complexes (18), whereas in the majority of active complexes transcript cleavage caused upstream shifting of the enzyme (9, 19).Many studies of arrest were performed with artificially halted individual elongation complexes (ECs) obtained by ''walking'' the RNA...

Early Transcriptional Arrest at Escherichia coli rplN and ompX Promoters

“…These inactive/stalled TCs can be reactivated by the addition of Gre factors and may thus correspond to previously described dead end or arrested complexes (3,12,13,39). When analysis similar to the one described above was performed with several templates driven by well studied E. coli RNAP promoters such as T7 A1, rrnB P1, and T5 N25, no stalled or arrested promoter-proximal TCs that were sensitive to Gre factors could be detected (data not shown).…”

“…Inactive/Stalled Promoter-proximal TCs Formed on PrplN and PompX Are backtracked-Previously characterized arrested TCs were shown to form when the RNAP catalytic center disengages from the 3Ј end of the nascent transcript and the enzyme backslides along the DNA (12,13). The "extra" RNA proximal to the 3Ј end is likely accommodated in the RNAP secondary channel, which also accepts the transcript cleavage factors (28,40).…”

“…During transcription in vitro and in vivo, RNAP can become arrested upon encountering a roadblock such as a DNA-binding protein or a special DNA sequence (1,11). Some roadblocks induce RNAP to slide backwards along the template ("backtrack") (11)(12)(13)(14), forcing the nascent RNA 3Ј-terminus to extrude through the RNAP secondary channel, typically by 2-18 nt (1-4, 12, 13). Endonucleolytic cleavage of such extruded RNA by the RNAP catalytic center generates a new 3Ј terminus that can be extended in the presence of rNTPs, giving RNAP another chance to read through the roadblock (11)(12)(13)(14).…”

“…Some roadblocks induce RNAP to slide backwards along the template ("backtrack") (11)(12)(13)(14), forcing the nascent RNA 3Ј-terminus to extrude through the RNAP secondary channel, typically by 2-18 nt (1-4, 12, 13). Endonucleolytic cleavage of such extruded RNA by the RNAP catalytic center generates a new 3Ј terminus that can be extended in the presence of rNTPs, giving RNAP another chance to read through the roadblock (11)(12)(13)(14). RNA cleavage by RNAP alone is inefficient under physiological conditions but is markedly stimulated by Gre/SII or, in the absence of the factors, by alkaline pH or pyrophosphate (15)(16)(17).…”

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

“…Note that we have not formally excluded the possibility that interactions between RNA and polymerase may also contribute to the energy barrier to backward movement of the enzyme during backtracking; however, it has been shown that addition of oligonucleotides complimentary to regions of the nascent RNA attenuates backtracking and stabilizes active transcription (33). This observation runs contrary to what one would expect if the mechanism of pause attenuation were mediated through RNA/protein interactions, as hybridization with the oligonucleotides would presumably compete with the RNA/protein interactions.…”