A movie of RNA polymerase II transcription.

We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

Structure of the transcription coactivator SAGA.

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)1,2 and transcription factor IID (TFIID)2-4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7-9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10-14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15-17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.

A pilot integrated model nurse clinic increases the uptake of antiviral treatment for the prevention of mother-to-child transmission of HBV.

BACKGROUND AND AIMS: Mother-to-child-transmission (MTCT) of hepatitis B virus (HBV) may still occur despite birth-dose HBV vaccinations when pregnant women are positive for hepatitis B surface antigen (HBsAg) with high viral loads (HBV DNA ≥ 200 000 IU/mL). A pilot integrated model nurse clinic (IMNC) was started in 2020 to implement the pre-emptive antiviral therapy with tenofovir disoproxil fumarate (TDF). We aimed to evaluate the performance of IMNC on uptake of TDF. METHODS: This was a territory-wide retrospective cohort of all consecutive HBsAg-positive women of child-bearing age with pregnancy records in public hospitals 2019-2022. Demographic characteristics, liver biochemistries and virologic parameters, and TDF use were collected. Concurrently, data from a prospective audit in Union Hospital, the private hospital with the highest number of deliveries in Hong Kong, from June 2022 to May 2023 were compared. RESULTS: The prevalence rate of HBV DNA ≥ 200 000 IU/mL in pregnant women with available HBV DNA records was 29.2% (66/226) in 2019, 27.3% (99/363) in 2020, 15.9% (125/784) in 2021 and 17.2% (117/679) in 2022 (p < .001), out of 2052 pregnant women who had their HBV DNA checked within 1 year prior to delivery. An increasing uptake rate of TDF by highly viraemic pregnant women (i.e. ≥ 200 000 IU/mL) was noted after the commencement of IMNC in public hospitals, with 67% (45/67) in 2019, 83% (88/106) in 2020, 91% (117/128) in 2021 and 89% (149/167) in 2022. Moreover, all highly viraemic pregnant women from Union Hospital received TDF. Continuous use of TDF was associated with a reduced risk of postpartum biochemical flare. CONCLUSIONS: IMNC increases the uptake of antiviral treatment in pregnant women at risk of MTCT of HBV. IMNC contributes to hepatitis elimination through a structured care plan to prevent MTCT of HBV.

Effective Components of Social Emotional Learning Programs: A Meta-analysis.

Previous reviews have synthesized the impacts of universal school-based social emotional learning (SEL) programs. However, they have yet to attempt a meta-analytic approach with rigorous inclusion criteria to identify the key SEL components and explore what make these programs work. This study aims to fill that gap by examining the impacts of SEL programs and exploring the moderating effects of methodological characteristics, implementation features, and program components on SEL effectiveness. The final sample consisted of 12 high-quality SEL programs, 59 studies, and 83,233 participants, with an overall effect size of 0.15. Meta-regression results indicated that these SEL programs could significantly improve youth social emotional skills, reinforce affect and attitudes, promote academic performance, increase prosocial behaviors, and reduce antisocial behaviors. Training teachers' social emotional skills and reducing cognitive elements in SEL curricula were found to be effective components of SEL programs, whereas pedagogical activities, climate support, and family engagement were not. Large-scale studies of SEL programs tended to generate smaller effect sizes, and those with low program dosages were found to be less effective than those approaching the recommended dosage. Policy and practical implications on how to scale SEL programs are discussed.

Understanding secondary students' continuance intention to adopt AI-powered intelligent tutoring system for English learning.

Previous studies have demonstrated the effectiveness of intelligent tutoring systems (ITS) in facilitating English learning. However, no empirical research has been conducted on secondary students' intention to use ITSs in the language domain. This study proposes an extended technology acceptance model (TAM) to predict secondary students' continuance intention to use and actual use of ITSs for English learning. The model included fifteen hypotheses that were tested with 528 senior secondary students in China. The results of structural equation modeling showed that (1) perceived usefulness and price value had direct positive impacts on continuance intention; (2) perceived ease of use was not directly associated with students' intention but indirectly influenced intention via perceived usefulness; (3) through the mediation of perceptions, learning goal orientation and facilitating conditions were positively associated with continuance intention; (4) perceived enjoyment positively predicted and anxiety negatively predicted students' intention to use ITSs; and (5) students' continuance intention to use ITSs was significantly positively associated with their actual use of ITSs for English learning. The model showed strong explanatory power and might be implemented in future research. This study contributes to the theory and practice of ITSs in K-12 education.

Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage.

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.

Development of a data-driven COVID-19 prognostication tool to inform triage and step-down care for hospitalised patients in Hong Kong: a population-based cohort study.

BACKGROUND: This is the first study on prognostication in an entire cohort of laboratory-confirmed COVID-19 patients in the city of Hong Kong. Prognostic tool is essential in the contingency response for the next wave of outbreak. This study aims to develop prognostic models to predict COVID-19 patients' clinical outcome on day 1 and day 5 of hospital admission. METHODS: We did a retrospective analysis of a complete cohort of 1037 COVID-19 laboratory-confirmed patients in Hong Kong as of 30 April 2020, who were admitted to 16 public hospitals with their data sourced from an integrated electronic health records system. It covered demographic information, chronic disease(s) history, presenting symptoms as well as the worst clinical condition status, biomarkers' readings and Ct value of PCR tests on Day-1 and Day-5 of admission. The study subjects were randomly split into training and testing datasets in a 8:2 ratio. Extreme Gradient Boosting (XGBoost) model was used to classify the training data into three disease severity groups on Day-1 and Day-5. RESULTS: The 1037 patients had a mean age of 37.8 (SD ± 17.8), 53.8% of them were male. They were grouped under three disease outcome: 4.8% critical/serious, 46.8% stable and 48.4% satisfactory. Under the full models, 30 indicators on Day-1 and Day-5 were used to predict the patients' disease outcome and achieved an accuracy rate of 92.3% and 99.5%. With a trade-off between practical application and predictive accuracy, the full models were reduced into simpler models with seven common specific predictors, including the worst clinical condition status (4-level), age group, and five biomarkers, namely, CRP, LDH, platelet, neutrophil/lymphocyte ratio and albumin/globulin ratio. Day-1 model's accuracy rate, macro-/micro-averaged sensitivity and specificity were 91.3%, 84.9%/91.3% and 96.0%/95.7% respectively, as compared to 94.2%, 95.9%/94.2% and 97.8%/97.1% under Day-5 model. CONCLUSIONS: Both Day-1 and Day-5 models can accurately predict the disease severity. Relevant clinical management could be planned according to the predicted patients' outcome. The model is transformed into a simple online calculator to provide convenient clinical reference tools at the point of care, with an aim to inform clinical decision on triage and step-down care.

RNA polymerase I structure and transcription regulation.

Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An 'expander' element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A 'connector' element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase 'core' and 'shelf' modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core-shelf interface that includes a 'composite' active site for RNA chain initiation, elongation, proofreading and termination.

Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth.

Cell growth is regulated during RNA polymerase (Pol) I transcription initiation by the conserved factor Rrn3/TIF-IA in yeast/humans. Here we provide a structure-function analysis of Rrn3 based on a combination of structural biology with in vivo and in vitro functional assays. The Rrn3 crystal structure reveals a unique HEAT repeat fold and a surface serine patch. Phosphorylation of this patch represses human Pol I transcription, and a phospho-mimetic patch mutation prevents Rrn3 binding to Pol I in vitro and reduces cell growth and Pol I gene occupancy in vivo. Cross-linking indicates that Rrn3 binds Pol I between its subcomplexes, AC40/19 and A14/43, which faces the serine patch. The corresponding region of Pol II binds the Mediator head that cooperates with transcription factor (TF) IIB. Consistent with this, the Rrn3-binding factor Rrn7 is predicted to be a TFIIB homolog. This reveals the molecular basis of Rrn3-regulated Pol I initiation and cell growth, and indicates a general architecture of eukaryotic transcription initiation complexes.

Structural basis of RNA polymerase II backtracking, arrest and reactivation.

During gene transcription, RNA polymerase (Pol) II moves forwards along DNA and synthesizes messenger RNA. However, at certain DNA sequences, Pol II moves backwards, and such backtracking can arrest transcription. Arrested Pol II is reactivated by transcription factor IIS (TFIIS), which induces RNA cleavage that is required for cell viability. Pol II arrest and reactivation are involved in transcription through nucleosomes and in promoter-proximal gene regulation. Here we present X-ray structures at 3.3 Å resolution of an arrested Saccharomyces cerevisiae Pol II complex with DNA and RNA, and of a reactivation intermediate that additionally contains TFIIS. In the arrested complex, eight nucleotides of backtracked RNA bind a conserved 'backtrack site' in the Pol II pore and funnel, trapping the active centre trigger loop and inhibiting mRNA elongation. In the reactivation intermediate, TFIIS locks the trigger loop away from backtracked RNA, displaces RNA from the backtrack site, and complements the polymerase active site with a basic and two acidic residues that may catalyse proton transfers during RNA cleavage. The active site is demarcated from the backtrack site by a 'gating tyrosine' residue that probably delimits backtracking. These results establish the structural basis of Pol II backtracking, arrest and reactivation, and provide a framework for analysing gene regulation during transcription elongation.

Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.

We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.

A model for transcription initiation in human mitochondria.

Regulation of transcription of mtDNA is thought to be crucial for maintenance of redox potential and vitality of the cell but is poorly understood at the molecular level. In this study we mapped the binding sites of the core transcription initiation factors TFAM and TFB2M on human mitochondrial RNA polymerase, and interactions of the latter with promoter DNA. This allowed us to construct a detailed structural model, which displays a remarkable level of interaction between the components of the initiation complex (IC). The architecture of the mitochondrial IC suggests mechanisms of promoter binding and recognition that are distinct from the mechanisms found in RNAPs operating in all domains of life, and illuminates strategies of transcription regulation developed at the very early stages of evolution of gene expression.

A novel intermediate in transcription initiation by human mitochondrial RNA polymerase.

The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation-TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate-a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein-protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.

Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

Structural basis of initial RNA polymerase II transcription.

During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II-DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site. The ITC-mimicking structures show that two invariant lysine residues anchor the 3'-proximal phosphate of short RNAs. Short DNA-RNA hybrids adopt a tilted conformation that excludes the +1 template nt from the active site. NTP binding induces complete DNA translocation and the standard hybrid conformation. Conserved NTP contacts indicate a universal mechanism of NTP selection. The essential residue Q1078 in the closed trigger loop binds the NTP 2'-OH group, explaining how the trigger loop couples catalysis to NTP selection, suppressing dNTP binding and DNA synthesis.

Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.

Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

The RNA polymerase trigger loop functions in all three phases of the transcription cycle.

The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA-RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2'-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.

Effects of salient factors on the pursuit of higher education among multicultural youth in Hong Kong.

This study presents novel and compelling evidence of the disparities in factors influencing the beliefs and aspirations for higher education among mainstream and immigrant youth in Hong Kong, particularly those who are underprivileged. We developed and validated a psychometric questionnaire, known as the Post-Secondary Education Pursuit Instrument (PSEPi), which was administered to 4,850 students aged between 15 and 18 years old from 23 secondary schools. The objective of this study was to explore the factors that impact students' choices and plans for higher education. The results of the one-way Multivariate Analysis of Variance (MANOVA) analysis deepen our understanding of the differential effects of success and obstacle factors on students' higher education pursuits across various cultural groups. The underprivileged mainstream, Chinese immigrant, and ethnic minority South Asian youth reported a perceived glass ceiling effect associated with their ethnic backgrounds, as well as financial aid barriers, while pursuing higher education, in contrast to their average Hong Kong mainstream counterparts (mean Cohen's d = 0.40). The direct effects and multiple mediation analyses demonstrated that significant others' influence, particularly the influence of parents, and locus of control are prime determinants of the perceived usefulness of higher education for all student groups. The implications are that educational policies should be implemented to level the playing field in higher education admissions for both privileged and underprivileged youth in Hong Kong and other international countries. Overall, this study provides robust empirical evidence that can be utilized to enhance educational policies and practices to bridge the gap between mainstream and underprivileged immigrant youth in their pursuit of higher education.

Comparative antibody and cell-mediated immune responses, reactogenicity, and efficacy of homologous and heterologous boosting with CoronaVac and BNT162b2 (Cobovax): an open-label, randomised trial.

BACKGROUND: Few trials have compared homologous and heterologous third doses of COVID-19 vaccination with inactivated vaccines and mRNA vaccines. The aim of this study was to assess immune responses, safety, and efficacy against SARS-CoV-2 infection following homologous or heterologous third-dose COVID-19 vaccination with either one dose of CoronaVac (Sinovac Biotech; inactivated vaccine) or BNT162b2 (Fosun Pharma-BioNTech; mRNA vaccine). METHODS: This is an ongoing, randomised, allocation-concealed, open-label, comparator-controlled trial in adults aged 18 years or older enrolled from the community in Hong Kong, who had received two doses of CoronaVac or BNT162b2 at least 6 months earlier. Participants were randomly assigned, using a computer-generated sequence, in a 1:1 ratio with allocation concealment to receive a (third) dose of CoronaVac or BNT162b2 (ancestral virus strain), stratified by types of previous COVID-19 vaccination (homologous two doses of CoronaVac or BNT162b2). Participants were unmasked to group allocation after vaccination. The primary endpoint was serum neutralising antibodies against the ancestral virus at day 28 after vaccination in each group, measured as plaque reduction neutralisation test (PRNT50) geometric mean titre (GMT). Surrogate virus neutralisation test (sVNT) mean inhibition percentage and PRNT50 titres against omicron BA.1 and BA.2 subvariants were also measured. Secondary endpoints included geometric mean fold rise (GMFR) in antibody titres; incidence of solicited local and systemic adverse events; IFNγ+ CD4+ and IFNγ+ CD8+ T-cell responses at days 7 and 28; and incidence of COVID-19. Within-group comparisons of boost in immunogenicity from baseline and between-group comparisons were done according to intervention received (ie, per protocol) by paired and unpaired t test, respectively, and cumulative incidence of infection was compared using Kaplan-Meier curves and a proportional hazards model to estimate hazard ratio. The trial is registered with ClinicalTrials.gov, NCT05057169. FINDINGS: We enrolled participants from Nov 12, 2021, to Jan 27, 2022. We vaccinated 219 participants who previously received two doses of CoronaVac, including 101 randomly assigned to receive CoronaVac (CC-C) and 118 randomly assigned to receive BNT162b2 (CC-B) as their third dose; and 232 participants who previously received two doses of BNT162b2, including 118 randomly assigned to receive CoronaVac (BB-C) and 114 randomly assigned to receive BNT162b2 (BB-B) as their third dose. The PRNT50 GMTs on day 28 against ancestral virus were 109, 905, 92, and 816; against omicron BA.1 were 9, 75, 8, and 86; and against omicron BA.2 were 6, 80, 6, and 67 in the CC-C, CC-B, BB-C, and BB-B groups, respectively. Mean sVNT inhibition percentages on day 28 against ancestral virus were 83%, 96%, 87%, and 96%; against omicron BA.1 were 15%, 58%, 19%, and 69%; and against omicron BA.2 were 43%, 85%, 50%, and 90%, in the CC-C, CC-B, BB-C, and BB-B groups, respectively. Participants who had previously received two doses of CoronaVac and a BNT162b2 third dose had a GMFR of 12 (p<0·0001) compared with those who received a CoronaVac third dose; similarly, those who had received two doses of BNT162b2 and a BNT162b2 third dose had a GMFR of 8 (p<0·0001). No differences in CD4+ and CD8+ T-cell responses were observed between groups. We did not identify any vaccination-related hospitalisation within 1 month after vaccination. We identified 58 infections when omicron BA.2 was predominantly circulating, with cumulative incidence of 15·3% and 15·4% in the CC-C and CC-B groups, respectively (p=0·93), and 16·7% and 14·0% in the BB-C and BB-B groups, respectively (p=0·56). INTERPRETATION: Similar levels of incidence of, presumably, omicron BA.2 infections were observed in each group despite very weak antibody responses to BA.2 in the recipients of a CoronaVac third dose. Further research is warranted to identify appropriate correlates of protection for inactivated COVID-19 vaccines. FUNDING: Health and Medical Research Fund, Hong Kong. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.