Assessment of the nucleotide modifications in the high-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit.

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible

Nucleic Acids Res

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3'-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5'- and 3'-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field

Nucleic Acids Res

Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25 degrees C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase'). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices ('transitional phase'). After 2 h from the beginning (about 8-10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes ('steady-state phase'). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes

Nucleic Acids Res

Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 A resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work

Abstract P-27: The 30S Ribosomal Subunit Assembly Factor Rbfa Plays a Key Role in the Formation of the Central Pseudoknot and in the Correct Docking of Helix 44 of the Decoding Center

Background: Ribosome biogenesis is a complicated multi-stage process. In the cell, 30S ribosomal subunit assembly is fast and efficient, proceeding with the help of numerous assembly protein factors. The exact role of most assembly factors and mechanistic details of their operation remain unclear. The combination of genetic modification with cryo-EM analysis is widely used to identify the role of protein factors in assisting specific steps of the ribosome assembly process. The strain with knockout of a single assembly factor gene accumulates immature ribosomal particles which structural characterization reveals the information about the reactions catalyzed by the corresponding factor. Methods: We isolated the immature 30S subunits (pre-30S subunits) from the Escherichia coli strain lacking the rbfA gene (ΔrbfA) and characterized them by cryo-electron microscopy (cryo-EM). Results: Deletion of the assembly factor RbfA caused a substantial distortion of the structure of an important central pseudoknot which connects three major domains of 30S subunit and is necessary for ribosome stability. It was shown that the relative order of the assembly of the 3′ head domain and the docking of the functionally important helix 44 depends on the presence of RbfA. The formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. The cryo-EM maps for pre-30S subunits were divided into the classes corresponding to consecutive assembly intermediates: from the particles with completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and with partially distorted helix 44. Cryo-EM analysis of ΔrbfA 30S particles revealing the accumulation of two predominant classes of early and late intermediates (obtained at 2.7 Å resolutions) allowed us to suggest that RbfA participate in two stages of the 30S subunit assembly and is deeper involved in the maturation process than previously thought. Conclusion: In summary, RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including the folding of the head, and positioning of helix 44 in the decoding center at a later stage. An update to the model of factor-dependent 30S maturation was proposed, suggesting that RfbA is involved in most of the subunit assembly process

Completion of the 8 MW Multi-Frequency ECRH System at ASDEX Upgrade

Over the last 15 years, the Electron Cyclotron Resonance Heating (ECRH) system at the ASDEX Upgrade tokamak has been upgraded from a 2 MW, 2 s, 140 GHz system to an 8 MW, 10 s, dual frequency system (105/140 GHz). Eight gyrotrons were in routine operation during the current experimental campaign. All gyrotrons are step-tunable operating at 105 and 140 GHz with a maximum output power of about 1 MW and 10 s pulse length. The system includes 8 transmission lines, mainly consisting of oversized corrugated waveguides (I.D. = 87 mm) with overall lengths between 50 and 70 meters including quasi-optical sections at both ends. Further improvements of the transmission lines with respect to power handling and reliability are underway

Melflufen or pomalidomide plus dexamethasone for patients with multiple myeloma refractory to lenalidomide (OCEAN): a randomised, head-to-head, open-label, phase 3 study

Background Melphalan flufenamide (melflufen), an alkylating peptide-drug conjugate, plus dexamethasone showed clinical activity and manageable safety in the phase 2 HORIZON study. We aimed to determine whether melflufen plus dexamethasone would provide a progression-free survival benefit compared with pomalidomide plus dexamethasone in patients with previously treated multiple myeloma. Methods In this randomised, open-label, head-to-head, phase 3 study (OCEAN), adult patients (aged ≥18 years) were recruited from 108 university hospitals, specialist hospitals, and community-based centres in 21 countries across Europe, North America, and Asia. Eligible patients had an ECOG performance status of 0–2; must have had relapsed or refractory multiple myeloma, refractory to lenalidomide (within 18 months of randomisation) and to the last line of therapy; and have received two to four previous lines of therapy (including lenalidomide and a proteasome inhibitor). Patients were randomly assigned (1:1), stratified by age, number of previous lines of therapy, and International Staging System score, to either 28-day cycles of melflufen and dexamethasone (melflufen group) or pomalidomide and dexamethasone (pomalidomide group). All patients received dexamethasone 40 mg orally on days 1, 8, 15, and 22 of each cycle. In the melflufen group, patients received melflufen 40 mg intravenously over 30 min on day 1 of each cycle and in the pomalidomide group, patients received pomalidomide 4 mg orally daily on days 1 to 21 of each cycle. The primary endpoint was progression-free survival assessed by an independent review committee in the intention-to-treat (ITT) population. Safety was assessed in patients who received at least one dose of study medication. This study is registered with ClinicalTrials.gov, NCT03151811, and is ongoing. Findings Between June 12, 2017, and Sept 3, 2020, 246 patients were randomly assigned to the melflufen group (median age 68 years [IQR 60–72]; 107 [43%] were female) and 249 to the pomalidomide group (median age 68 years [IQR 61–72]; 109 [44%] were female). 474 patients received at least one dose of study drug (melflufen group n=228; pomalidomide group n=246; safety population). Data cutoff was Feb 3, 2021. Median progression-free survival was 6·8 months (95% CI 5·0–8·5; 165 [67%] of 246 patients had an event) in the melflufen group and 4·9 months (4·2–5·7; 190 [76%] of 249 patients had an event) in the pomalidomide group (hazard ratio [HR] 0·79, [95% CI 0·64–0·98]; p=0·032), at a median follow-up of 15·5 months (IQR 9·4–22·8) in the melflufen group and 16·3 months (10·1–23·2) in the pomalidomide group. Median overall survival was 19·8 months (95% CI 15·1–25·6) at a median follow-up of 19·8 months (IQR 12·0–25·0) in the melflufen group and 25·0 months (95% CI 18·1–31·9) in the pomalidomide group at a median follow-up of 18·6 months (IQR 11·8–23·7; HR 1·10 [95% CI 0·85–1·44]; p=0·47). The most common grade 3 or 4 treatment-emergent adverse events were thrombocytopenia (143 [63%] of 228 in the melflufen group vs 26 [11%] of 246 in the pomalidomide group), neutropenia (123 [54%] vs 102 [41%]), and anaemia (97 [43%] vs 44 [18%]). Serious treatment-emergent adverse events occurred in 95 (42%) patients in the melflufen group and 113 (46%) in the pomalidomide group, the most common of which were pneumonia (13 [6%] vs 21 [9%]), COVID-19 pneumonia (11 [5%] vs nine [4%]), and thrombocytopenia (nine [4%] vs three [1%]). 27 [12%] patients in the melflufen group and 32 [13%] in the pomalidomide group had fatal treatment-emergent adverse events. Fatal treatment-emergent adverse events were considered possibly treatment related in two patients in the melflufen group (one with acute myeloid leukaemia, one with pancytopenia and acute cardiac failure) and four patients in the pomalidomide group (two patients with pneumonia, one with myelodysplastic syndromes, one with COVID-19 pneumonia). Interpretation Melflufen plus dexamethasone showed superior progression-free survival than pomalidomide plus dexamethasone in patients with relapsed or refractory multiple myeloma.Oncopeptides ABPeer reviewe