Molecular Cell

Stress granules (SGs) are cytoplasmic biomolecular condensates that assemble when translation initiation stalls, sequestering stalled preinitiation complexes and associated RNA-binding proteins. How individual initiation factors are targeted to SGs and released following stress recovery to reinitiate translation remains poorly understood. Here, combining a NEDD8-conjugate proteome with our previously reported arsenite-induced NEDD8 interactome and curated RNA granule databases, we find that eIF3g and eIF3i are shared, high-confidence NEDDylated SG components. NEDDylation of eIF3g and eIF3i-associated complexes is readily detected at steady state and declines under arsenite stress. Intriguingly, only full-length eIF3g is recruited to SGs. eIF3g lacking the RRM domain strongly inhibits SG formation, whereas the RRM domain alone neither inhibits SG assembly nor localizes to SGs. Blocking the NEDD8 pathway--by NAE inhibition with MLN4924, depletion of NEDD8 pathway components, or expression of the deNEDDylase NEDP1--accelerates the loss of eIF3g and eIF3i protein during stress. Our data indicate that NEDDylation marks a degradation-resistant pool of eIF3g/eIF3i that is competent for SG localization, linking the NEDD8 pathway to initiation-factor proteostasis and condensate partitioning, and potentially making these factors available for translation reinitiation during stress recovery.

DNA double-strand breaks (DSBs) activate repair pathways that must be coordinated with other cellular functions. Although DSB sensors SIRT6, Ku80, and MRE11 initiate repair, how they organize these nuclear processes remains unknown. SPARK-ID is a proximity-labeling strategy mapping DDR interactome dynamics. Using these sensors as baits, we resolved chromatin-associated interactomes from damage formation to recovery. The sensors shared an enriched repair core while capturing distinct interactors, allowing temporal specialization: SIRT6ID was biased toward RNA and chromatin regulation, Ku80ID toward telomere-associated and translational programs, and MRE11ID toward recombination and DNA synthesis. Modularity analysis showed these functions are organized into modules linked by "connectors". Among them, Nucleolin linked DNA repair, RNA-metabolism, and nucleolar modules. Nucleolin depletion rewired DSB-sensor interactomes, altered repair-associated complex composition, expanded {gamma}H2AX domains, and reduced BRCA1, 53BP1, and phospho-ATM foci. Altogether, SPARK-ID reveals modular DSB-sensor interactomes whose robustness depends on connectors that integrate and constrain the DNA damage response. Graphical AbstractThree DSB sensors (MRE11, Ku80, SIRT6) orchestrate DNA repair through dynamic protein-protein interaction networks that expand upon DSB induction. Proximity labeling reveals distinct sensor interactomes that share a functional core of nuclear processes while incorporating sensor-specific interactors for pathway specialization. These networks contain connector nodes that coordinate multiple parallel nuclear functions, enabling functional diversification and conferring robustness to the DNA damage response. The sensors reshape their interactome structure and composition to integrate and constrain cellular responses through organized macromolecular complexes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/740882v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1bb112dorg.highwire.dtl.DTLVardef@64cc4dorg.highwire.dtl.DTLVardef@1a0e2d1org.highwire.dtl.DTLVardef@18b3e8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The human genome encodes several hundreds of ubiquitin E3 enzymes, most of which have poorly understood biological roles. In search for novel ubiquitin E3 enzymes involved in DNA damage tolerance, we identified RNF25 as a candidate to have a role in DNA replication stress tolerance. Under stress conditions, RNF25 translocates to the nucleus in a cGAS-dependent manner, and loss of RNF25 ubiquitin E3 activity leads to the accumulation of replication-dependent ssDNA gaps. Combining functional assays with with mass-spectrometry based proteomics approaches such as TULIP2, iPOND-MS and TurboID, we found that, mechanistically, RNF25 promotes the stability of the replication fork at Transcription-Replication Conflicts by the ubiquitin-mediated clearance of RAD18, mono-ubiquitinated PCNA, H2B-K120ub and RECQL, among others. Lack of RNF25 ubiquitin E3 activity promotes the occurrence of Transcription-Replication Conflicts and destabilizes reversed replication forks, enabling re-priming and accumulation of ssDNA gaps behind the replication fork, ultimately compromising genome integrity. Overall, RNF25 regulates DNA replication, transcription and translation in an ubiquitin-based goldilocks.

Assembly of eukaryotic large ribosomal subunits (LSU) requires coordinated structural and compositional transitions within pre-60S particles, yet the underlying mechanisms remain poorly understood. Here, we show that the DEAD-box helicase Drs1 promotes early maturation across distinct regions of the pre-60S particle. Loss of Drs1 function causes accumulation of co-transcriptional intermediates retaining SSU processome components, indicating that Drs1 promotes timely separation of nascent LSU precursors from the small-subunit assembly pathway. Cryo-EM analyses reveal both a redistribution toward early nucleolar maturation states upon loss of Drs1, including Nsa1-deficient intermediates, and a confinement of Drs1-associated particles to states preceding stable incorporation of 25S rRNA domain III. Drs1 directly engages Erb1 through its unstructured N-terminal extension, promoting stable assembly of the Nop7-Erb1-Ytm1 module associated with domain III maturation. CRAC analysis localizes Drs1 to spatially clustered sites spanning the 5.8S and 25S rRNAs, encompassing domains I-IV. Together, these findings support a model in which Drs1 couples stabilization of assembly-factors with pre-rRNA remodeling across the pre-60S particle, thereby driving ordered early LSU maturation and the timed integration of domain III.

The coding potential of mature mRNAs is generally considered fixed once transcription and RNA processing are complete. We previously established RNA dicing as a post-transcriptional process that generates stable, uncapped, translation-competent RNA isoforms. Here, we identify RNA dicing as a transient post-transcriptional program that remodels mature transcripts during macrophage polarization. Long-read transcriptomics reveals widespread, fate-specific dicing that peaks during early cell-state transitions and preferentially occurs between protein domains, preserving downstream coding modules. Fractionated proteomics links these RNA isoforms to truncated protein products, indicating that dicing reshapes proteomic output. Using JAK1 as a mechanistic model, we show that disruption of dicing, impairs macrophage polarization towards pro-inflammatory states. Mechanistically, a diced JH1 kinase module displayed distinct substrate preferences and alters downstream signaling relative to full-length JAK1. These findings establish RNA dicing as an adaptive layer of gene regulation that reprograms transcript architecture, expands protein functional diversity, and helps shape cell-state transitions.

Cells respond to environmental stress by rapidly remodeling translation and assembling stress granules (SGs), which are dynamic ribonucleoprotein condensates that contain untranslated mRNAs, translation initiation factors, and 40S ribosomal subunits. Although the translation initiation factor eIF4B has been implicated in SG biology, the contribution of its highly conserved RNA recognition motif (RRM) to SG assembly has remained unclear. Here, we developed a quantitative live-cell imaging framework that resolves distinct kinetic phases of SG assembly at single-cell resolution and combines these measurements with single-cell analysis of protein synthesis. Using this approach, we show that disruption of the eIF4B RRM delays SG nucleation, slows SG assembly, and reduces the number of SGs formed, while having little effect on mature SG size. Biochemical analyses revealed that the RRM mutant retained high-affinity binding to both RNA and the 40S ribosomal subunit and exhibited only a modest reduction in eIF4A helicase stimulation activity but displayed altered RNA engagement, consistent with impaired RNA-dependent organization of the translation initiation machinery. Coupling SG kinetics with single-cell measurements of protein synthesis further revealed that delayed SG nucleation is associated with reduced translational repression during oxidative stress. Together, our findings identify the conserved eIF4B RRM as a regulator of productive higher-order organization of the translation initiation machinery and establish a quantitative framework for investigating how SG assembly and translational remodeling are coordinated during cellular stress.

NF-{kappa}B must remain bound to chromatin long enough to activate innate immune genes, but what stabilizes this transcriptionally productive state is unknown. Here, we identify the Integrator complex as an essential determinant of NF-{kappa}B chromatin residence in mouse and human cells. Integrator associated with NF-{kappa}B and co-occupied its genomic targets. Single-molecule tracking revealed that Integrator markedly prolonged the chromatin residence of RELA molecules in a low-mobility state. Catalytically inactive INTS11-E203Q fully restored NF-{kappa}B-dependent transcription, separating this chromatin-retention function from RNA cleavage. Conditional Ints11 deletion abolished TLR4-dependent dendritic-cell activation in adult mice and impaired LPS-induced transcription in mouse embryonic fibroblasts. Loss of Integrator in human cancer cells similarly suppressed TNF-induced inflammatory gene expression. These findings uncover a noncatalytic function for Integrator that stabilizes NF-{kappa}B at enhancers, complementing its established catalytic control of RNA polymerase II pause release. By coupling signal recognition at enhancers to productive elongation, Integrator provides a molecular solution to a central challenge of multicellularity: converting transient extracellular cues into coherent gene-expression programs across complex chromatin landscapes.

Maintenance of genome integrity requires accurate repair of DNA double-strand breaks (DSBs), particularly within transcriptionally active regions. Persistent R-loops at DSBs can impede homologous recombination (HR) repair. While factors that resolve R-loops at DSB sites are known, the mechanisms ensuring timely degradation of nascent RNA to prevent pathological R-loop accumulation remain elusive. Here, we identified a critical role for the RNA-binding protein LSm4 in orchestrating localized RNA decay at DSBs to facilitate repair. We demonstrated that among LSm1-8 subunits, only LSm4 undergoes liquid-liquid phase separation (LLPS) and forms biomolecular condensates (BCs) specifically at DSBs in transcriptionally active chromatin. These damage-induced LSm4 BCs function as hubs that promote nuclear RNA decapping and recruit the 5'[->]3' exonuclease XRN2 to degrade nascent transcripts proximal to DSBs. Accordingly, LSm4-XRN2 axis suppresses R-loop hyperaccumulation, thereby enabling efficient RAD51 filament assembly and intact HR repair. Consequently, loss of LSm4 increases translocations and leads to genomic instability. Collectively, our findings define a new regulatory layer in which LSm4 BCs spatially license RNA degradation, preventing R-loop accumulation at DSB microenvironment to facilitate error-free repair.

CRISPR adaptation requires Cas1-Cas2 to capture prespacers, undergo conformational rearrangement and catalyse integration, but how these steps are coupled remains unclear. Using nine hyperactive Escherichia coli Cas1 substitutions as perturbational probes, we identified a prespacer-coupling module intersecting an interior conformational-coupling module. Single substitutions increased adaptation up to sixfold, whereas combinatorial reassortment generated a genotype with 103-fold greater activity than wild type. Genotype-network analysis and quantitative reconstruction revealed strong background-dependent epistasis: the same substitution could enhance activity in one genotype but impair it in another, and high activity emerged only from compatible combinations spanning both modules. These findings indicate that Cas1-Cas2 activity is constrained by compatibility among changes distributed across the Cas1 dimer, rather than by optimization of individual catalytic or DNA-binding interactions. We propose that coordinated tuning of prespacer engagement and conformational coupling governs Cas1-Cas2 activity during CRISPR adaptation.

Gene fusions involving the FET gene family (FUS, EWSR1, and TAF15) act as drivers of numerous cancer subtypes. The resulting chimeric proteins are widely viewed as aberrant transcriptional regulators that promote malignant transformation through chromatin and enhancer reprogramming. Here, we show that FET fusion oncoproteins also function as regulators of alternative splicing across multiple sarcoma subtypes. Transcriptomic analyses revealed extensive but largely non-overlapping splicing programs driven by the EWSR1::FLI1, EWSR1::WT1, EWSR1::ATF1 and FUS::DDIT3 fusions that nevertheless converged on common oncogenic functions. Fusion-dependent splicing regulation was mechanistically separable from canonical transcriptional activity and was associated with extensive remodeling of cooperative RNA-binding protein (RBP) assemblies on target transcripts. Despite regulating distinct exons, different FET fusions engaged highly similar RBP interaction networks, consistent with a conserved mode of splicing regulation. Transcriptome-wide mapping of RBP occupancy revealed extensive reorganization of local RNA regulatory landscapes following fusion depletion. The requirement of RNA for FET fusion condensate formation, together with the inability of condensation-defective mutants to restore splicing regulation, further supported a role for higher-order assemblies in fusion-dependent alternative splicing (AS) control. Fusion-driven splicing programs stratified Ewing sarcoma patients independently of established clinical covariates, thereby underscoring their clinical relevance. AS of TFDP1 emerged as a common fusion-regulated splicing event required for sarcoma cell fitness and therapeutically actionable using antisense oligonucleotides. Together, our findings establish AS regulation as a conserved function of FET fusion oncoproteins that is mechanistically separable from their canonical transcriptional activity. More broadly, they support a model in which oncogenic fusion proteins can drive malignant phenotypes through large-scale remodeling of RNA regulatory networks.

Epigenetic information is transmitted through covalent DNA modifications that regulate chromatin structure and gene expression. 5-Hydroxymethylcytosine (5hmC) is a key epigenetic intermediate generated during TET-mediated oxidative demethylation of 5-methylcytosine. Whether 5hmC itself is intrinsically genotoxic remains unclear. Here, we combine genome-wide CRISPR loss-of-function screens, isogenic knockouts and mass spectrometry to systematically compare the cellular consequences of exogenous 5-hydroxymethyl-2'-cytidine (5hm-dC) exposure and endogenous oxidative demethylation in mammalian cells. We find that exogenous 5hm-dC causes toxicity, mediated by deamination to 5-hydroxymethyl-2'-deoxyuridine (5hm-dU), followed by excision by the glycosylase SMUG1, which generates base excision repair (BER) intermediates that compromise cell viability. In contrast, toxicity associated with enzymatic oxidative demethylation is primarily driven by TDG-dependent excision of oxidized methylcytosine derivatives. Despite these distinct initiating events, both genotoxins converge on a critical requirement for DNA polymerase {beta} (POL{beta}), indicating that efficient BER completion is essential to mitigate cytotoxic repair intermediates.

Eukaryotic RNA polymerase II transcripts carry a signature m7G cap (Cap0) structure that is co-transcriptionally added to the 5 end, and is essential for translation and RNA stability. Higher eukaryotes carry additional essential ribose methylations on the first and second cap-proximal nucleotides, termed as Cap1 and Cap2, respectively. The ubiquitous Cap1 modification protects cellular RNAs from being recognized by the innate immune sensors. Cap2 is also implicated in such an innate immune role, but here we use our genetic analyses of two human cell lines and three mouse tissues to reveal that loss of CMTR2 does not result in activation of the innate immune response. Germline deletion of mouse CMTR2 shows that it is required for male and female fertility. While mutant germ cells proceed into the meiotic pachytene spermatocyte stage, their transcriptome fails to keep pace and transition from the preceding leptotene/zygotene stages. Such a meiotic role is not conserved in other vertebrates like zebrafish, as cmtr2 mutants are fertile, instead it has a role in defining sex, as all mutants are exclusively males. Taken together, our study reveals that CMTR2 does not influence innate immune response in human cells and mouse tissues, but shapes gene expression during developmental transitions.

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.

The metallo-{beta}-lactamase (MBL)-domain superfamily includes multiple nucleases that process distinct transcript classes. We show that human MBLAC2 acts as a ribosomal (r)RNA quality control factor, being nucleolar specific, chromatin-associated and required for co-transcriptional pre-rRNA processing. Notably MBLAC2 is functionally distinct from other MBL containing proteins, being required for maintenance of nucleolar architecture, cell cycle progression and proliferative fitness. Selective MBLAC2 depletion causes accumulation of pre-rRNA intermediates that lead to perturbed RNA polymerase (Pol) I-associated RNA homeostasis and nucleolar stress signalling. Nascent RNA profiling with long-read nanopore sequencing reveals extended and aberrant pre-rRNA species, consistent with defective processing and transcript release. Remarkably disruption of rRNA processing reduces global Pol II transcriptional output, even though the Pol II nuclear distribution remains unchanged across genomic compartments. Altogether, we define MBLAC2 as the nucleolar branch of an MBL-domain RNA surveillance network that couples rRNA quality control with transcriptional homeostasis.

DNA double-strand break (DSB) repair by homologous recombination entails the coordinated search for a homologous dsDNA molecule by two heterotypic Rad51-ssDNA filaments in eukaryotes. How homology search is regulated in cells remains largely unknown. Using genomic and molecular assays to track spatial chromatin organization and early recombination intermediates, we investigated the roles in homology search of two antagonistic regulators of Rad51-ssDNA filaments metabolism in S. cerevisiae: the Rad51 paralogs Rad55-Rad57 and the 3'-5' ssDNA translocase Srs2. Srs2 promoted the coordinated search between filaments on each DSB ends and inactivated homology search following homology identification. Rad55-Rad57 both stimulated the formation of Rad51-ssDNA filaments and protected them against disruption by Srs2. Together, Rad55-Rad57 and Srs2 enacted a structural proof-reading that resulted in stiffer Rad51-ssDNA filaments competent for genome-wide homology search. This work reveals multiple ways by which the control of Rad51-ssDNA filament metastability by the individual and joint activities of Rad55-Rad57 and Srs2 regulate homology search onset, coordination, reach and inactivation.

Haem-Regulated Inhibitor (HRI, aka EIF2AK1) is one of four stress-sensing kinases that phosphorylate eIF2 as part of the integrated stress response (ISR). HRI was first characterized as a haem-sensing kinase where it is inhibited when bound to haem. Recent studies have revealed another role for HRI in sensing mitochondrial stress via the mitochondrial protein DELE1. Upon stress, DELE1 is cleaved, and its C-terminal fragment (DELE1CTD) is released from the mitochondria to the cytoplasm, where it interacts with HRI, to trigger the ISR. This pathway is critical for mitochondrial quality control and neuronal health. Here, we perform biophysical analysis to demonstrate that purified recombinant DELE1CTD binds with high affinity to the N-terminal region of HRI, competing with and displacing haem to activate HRI and pointing to a shared binding site. Using Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), we map the binding footprint of DELE1CTD on HRI and confirm that it overlaps with the haem-binding site. These findings support a simple activation mechanism: DELE1CTD activates HRI by excluding inhibitory haem from its binding site.

Faithful genome duplication requires coordination of DNA replication, chromatin assembly, and checkpoint signaling to prevent inappropriate mitotic progression. Using a targeted siRNA screen for factors modulating CHK1 inhibitor-induced DNA damage and premature mitotic entry, we identified replication-coupled chromatin assembly as a determinant of checkpoint dependence. Acute depletion of the CAF-1 subunit CHAF1A modestly delayed S-phase progression but caused little DNA damage, whereas inhibition of CHK1 or ATR triggered extensive DNA damage, premature mitotic entry, and loss of viability. Transcriptomic analyses revealed that CHK1 inhibition induced a transcriptional response enriched for B-MYB/Myb-MuvB (MMB) target genes in CHAF1A-deficient cells. Notably, induction of these genes occurred without corresponding increases in promoter accessibility, suggesting that enhanced MMB-dependent transcription is largely independent of widespread promoter opening. B-MYB depletion attenuated MMB target-gene induction, premature mitotic entry, and DNA damage. Depletion of ASF1 and NAP1L1 similarly enhanced checkpoint inhibitor sensitivity, indicating that increased dependence on ATR-CHK1 signaling is a broader consequence of impaired histone chaperone function. Together, our findings establish a functional link between replication-coupled chromatin assembly, ATR-CHK1 signaling, and MMB-dependent mitotic transcription.

Temozolomide (TMZ) is a frontline alkylating chemotherapy, yet its direct impact on RNA modification and global translation dynamics remains poorly understood. Here, we demonstrate that TMZ induces pervasive RNA alkylation causing severe translational impairment. TMZ directly deposits aberrant methyl groups onto single-stranded mRNA in vitro, creating physical lesions that lower translational efficiency. In glioblastoma cells, acute TMZ exposure triggers a rapid, widespread accumulation of m7G on cellular RNAs, leading to the significant attenuation of global protein synthesis. Nanopore direct RNA sequencing identified distinct guanine-specific error signatures and sequence context preferences associated with TMZ-induced damage. Using a quantitative yeast spike-in ribosome profiling strategy, we mapped this translational repression at transcript-level, revealing a global downregulation of translational efficiency. This widespread repression disproportionately targets highly interconnected networks essential for cellular proliferation, specifically chromosome organization. We show that the severity of this translational repression is driven by a transcript's coding guanine density, stability and translation initiation speed. Together, our findings suggest that TMZ-induced alkylation targets stable, highly translated, guanine-rich transcripts. This establishes aberrant RNA methylation and subsequent translational arrest as a potential mechanism of temozolomide cytotoxicity.

Androgen receptor-interacting proteins (AR-IPs) comprise nearly 1,000 cataloged partners, yet how AR engages this interactome inside the nucleus, in what temporal order, and through what molecular handoffs, remains uncharted. Here, we construct a minute-scale temporal atlas of the nuclear AR proximal interactome by proximity-labeling quantitative mass spectrometry (PL-qMS) in androgen-treated LNCaP prostate cancer cells, capturing 84.2% of the known AR-interactome and resolving 3,378 nuclear AR-proximal interacting proteins (AR-PIPs) across six time points. The atlas recapitulates the cyclic sequential recruitment model established at AR-regulated loci by classical ChIP and recovers 100% of previously known AR interactors from the Launonen 2021 ChIP-SICAP chromatome. Embedded within this canonical chromatin signature, we uncover a time-resolved translation-to-transcription handoff in which cap-binding eIF4G and 4E-BP1 are transiently AR-proximal at the earliest time points, verified by PLA. The atlas converts the AR coregulator catalog into a temporally resolved quantitative framework for AR-dependent transcription. SynopsisA minute-scale temporal atlas of the nuclear AR-proximal interactome resolves a translation-to-transcription handoff during the androgen response. O_LITemporal nuclear AR-proximal interactome atlas in androgen-treated LNCaP cells C_LIO_LI3,378 nuclear AR-PIPs recovered, 100% of previously known Launonen AR partners C_LIO_LICap-binding eIF4G and 4E-BP1 are transiently AR-proximal at early time points C_LIO_LIPLA verifies AR-eIF4G and AR-4E-BP1 proximal interactions pointing to a translation-to-transcription handoff C_LI

Eukaryotic transcription factors recognize short motifs, creating abundant binding sites within gene bodies and potential collisions with elongating RNA polymerases, yet how such encounters are resolved remains unclear. Here, we use optical tweezers to monitor RNA polymerase transcription through DNA-bound Egr-1, a zinc-finger transcription factor. Using DNA-fluctuation suppression as a readout of polymerase arrival, we show that Egr-1 delays elongation in an orientation- and rNTP-dependent manner, whereas force measurements indicate that RNAP does not bypass the TF by mechanical eviction. Instead, RNAP destabilizes the Egr-1-DNA complex over a short, structured interaction zone, increasing TF dissociation non-monotonically with distance. Monte Carlo simulations incorporating these kinetic changes recapitulate passage-time distributions. CpG methylation shortens Egr-1 residence time and largely eliminates the TF-dependent delay, suggesting a role for gene-body methylation in reducing kinetic barriers to elongation. These results reveal DNA-bound TFs as tunable barriers that locally shape transcription elongation.