The dynamic machinery of mRNA elongation

Armache, Karim‐Jean; Kettenberger, Hubert; Cramer, Patrick

doi:10.1016/j.sbi.2005.03.002

Cited by 17 publications

(14 citation statements)

References 37 publications

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“…In polymerases, M 2 is dynamic: it enters the active site with the incoming nucleotide and escorts the pyrophosphate product out of the active site after the reaction has occurred (34,35). This movement coincides with the power stroke that drives each round of polymerization.…”

Section: Discussionmentioning

confidence: 99%

A relaxed active site after exon ligation by the group I intron

Lipchock

Strobel

2008

Proc. Natl. Acad. Sci. U.S.A.

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During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.crystallography ͉ metalloenzyme ͉ ribozyme ͉ splicing G roup I intron splicing proceeds through two consecutive transesterification reactions separated by a conformational change. The first step proceeds by the addition of an exogenous G (␣G) to the 5Ј end of the intron. This covalently releases the 5Ј exon, but the exon remains bound through base-pairing and tertiary hydrogen bonds (1, 2). A conformational change moves the ␣G out of the active site and exposes a portion of the internal guide sequence (IGS) that is complementary to the 3Ј exon (3). The IGS then base-pairs to both the 5Ј and 3Ј exons, orienting them for the second step of splicing (reaction state pre-2S). During exon ligation the 5Ј exon attacks the scissile phosphate between the last nucleotide of the intron (⍀G) and the 3Ј exon (reaction state post-2S). After ligation, the exons are released from the intron in at least two steps (4, 5). The tertiary contacts to the exon-IGS helix (P1-P10 or substrate helix) are disrupted, resulting in an open or undocked complex (6, 7), and then the substrate helix dissociates to release the ligated exons (8, 9).Recent crystal structures have provided a molecular view of a subset of the covalent and conformational states along the splicing pathway. Two structures of the Azoarcus sp. BH72 pre-tRNA Ile group I intron captured the intron bound to both exons before the second step of splicing (pre-2S) (10, 11). In the first structure (deoxy pre-2S), the ligation reaction was prevented by the inclusion of 2Ј-deoxyribose substitutions at ⍀G, an essential hydroxyl group, and at three other positions (10). The second structure (ribo pre-2S) contained a ribose at all positions, including ⍀G, except U-1 (the last nucleotide of the 5Ј exon) (11). The resulting complex had some activity in solution and within the crystals. Two other group I intron structures were reported soon after the Azoarcus intron. The firs...

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Section: Discussionmentioning

confidence: 99%

A relaxed active site after exon ligation by the group I intron

Lipchock

Strobel

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Consistent with the hypothesis that cancer EST heterogeneity is due to transcription infidelity is the finding that bases most influential of the substitution events are not only the substituted base itself (b0), but also the four bases located upstream and the three downstream of this event. This pattern corresponds in the elongation complex with the first four RNA-DNA base-pairing and to the three transcription-driven melted bases (23). In vitro studies of RNAP infidelity established that DNA grip three bases downstream of the misalignment event is critical at controlling transcription fidelity (24,25).…”

Section: Discussionmentioning

confidence: 99%

Nonrandom variations in human cancer ESTs indicate that mRNA heterogeneity increases during carcinogenesis

Brulliard¹,

Lorphelin²,

Collignon

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Virtually all cancer biological attributes are heterogeneous. Because of this, it is currently difficult to reconcile results of cancer transcriptome and proteome experiments. It is also established that cancer somatic mutations arise at rates higher than suspected, but yet are insufficient to explain all cancer cell heterogeneity. We have analyzed sequence variations of 17 abundantly expressed genes in a large set of human ESTs originating from either normal or cancer samples. We show that cancer ESTs have greater variations than normal ESTs for >70% of the tested genes. These variations cannot be explained by known and putative SNPs.

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“…Each mechano-chemical cycle of the RNAP during the elongation stage [13,52,53] consists of several steps; the major steps being (i) Nucleoside triphosphate (NTP) binding to the active site of the RNAP when the active site is located at the growing tip of the mRNA transcript, (ii) NTP hydrolysis, (iii) release of pyrophosphate (P P i ), one of the products of hydrolysis, and (iv) accompanying forward stepping of the RNAP [13]. This simplified scenario, which is adequete for our purpose here, is shown symbolically in equation (1):…”

Section: Brief Review Of Phenomenology and Earlier Modelsmentioning

confidence: 99%

Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

Tripathi

Chowdhury

2008

Phys. Rev. E

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RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two new measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

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The dynamic machinery of mRNA elongation

Cited by 17 publications

References 37 publications

A relaxed active site after exon ligation by the group I intron

A relaxed active site after exon ligation by the group I intron

Nonrandom variations in human cancer ESTs indicate that mRNA heterogeneity increases during carcinogenesis

Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

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