Volume 404, Issue 8-9

There is a growing interest in characterizing the structure and dynamics of large biomolecular assemblies and their interactions within the cellular environment. A diverse array of experimental techniques allows us to study biomolecular systems on a variety of length and time scales. These techniques range from imaging with light, X-rays or electrons, to spectroscopic methods, cross-linking mass spectrometry and functional genomics approaches, and are complemented by AI-assisted protein structure prediction methods. A challenge is to integrate all of these data into a model of the system and its functional dynamics. This review focuses on Bayesian approaches to integrative structure modeling. We sketch the principles of Bayesian inference, highlight recent applications to integrative modeling and conclude with a discussion of current challenges and future perspectives.

In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection.

Mitochondria are the essential players in eukaryotic ATP production by oxidative phosphorylation, which relies on the maintenance and accurate expression of the mitochondrial genome. Even though the basic principles of translation are conserved due to the descendance from a bacterial ancestor, some deviations regarding translation factors as well as mRNA characteristics and the applied genetic code are present in human mitochondria. Together, these features are certain challenges during translation the mitochondrion has to handle. Here, we discuss the current knowledge regarding mitochondrial translation focusing on the termination process and the associated quality control mechanisms. We describe how mtRF1a resembles bacterial RF1 mechanistically and summarize in vitro and recent in vivo data leading to the conclusion of mtRF1a being the major mitochondrial release factor. On the other hand, we discuss the ongoing debate about the function of the second codon-dependent mitochondrial release factor mtRF1 regarding its role as a specialized termination factor. Finally, we link defects in mitochondrial translation termination to the activation of mitochondrial rescue mechanisms highlighting the importance of ribosome-associated quality control for sufficient respiratory function and therefore for human health.

During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation of RNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.

Soluble nuclear transport receptors and stationary nucleoporins are at the heart of the nucleocytoplasmic transport machinery. A subset of nucleoporins contains characteristic and repetitive FG (phenylalanine-glycine) motifs, which are the basis for the permeability barrier of the nuclear pore complex (NPC) that controls transport of macromolecules between the nucleus and the cytoplasm. FG-motifs can interact with each other and/or with transport receptors, mediating their translocation across the NPC. The molecular details of homotypic and heterotypic FG-interactions have been analyzed at the structural level. In this review, we focus on the interactions of nucleoporins with nuclear transport receptors. Besides the conventional FG-motifs as interaction spots, a thorough structural analysis led us to identify additional similar motifs at the binding interface between nucleoporins and transport receptors. A detailed analysis of all known human nucleoporins revealed a large number of such phenylalanine-containing motifs that are not buried in the predicted 3D-structure of the respective protein but constitute part of the solvent-accessible surface area. Only nucleoporins that are rich in conventional FG-repeats are also enriched for these motifs. This additional layer of potential low-affinity binding sites on nucleoporins for transport receptors may have a strong impact on the interaction of transport complexes with the nuclear pore and, thus, the efficiency of nucleocytoplasmic transport.

Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes – TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.

Atg18, Atg21 and Hsv2 are homologous β-propeller proteins binding to PI3P and PI(3,5)P 2 . Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with both the ER and the vacuole. Atg21 is restricted to the vacuole phagophore contact, where it organizes part of the Atg8-lipidation machinery. The role of Hsv2 is less understood, it partly affects micronucleophagy. Atg18 is further involved in regulation of PI(3,5)P 2 synthesis. Recently, a novel Atg18-retromer complex and its role in vacuole homeostasis and membrane fission was uncovered.

The cytoskeleton of eukaryotes consists of actin filaments, microtubules and intermediate filaments (IF). IFs, in particular, are prone to pronounced phosphorylation, leading to additional charges on the affected amino acids. In recent years, a variety of experiments employing either reconstituted protein systems or living cells have revealed that these altered charge patterns form the basis for a number of very diverse cellular functions and processes, including reversible filament assembly, filament softening, network remodeling, cell migration, interactions with other protein structures, and biochemical signaling.

Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete structures of both yeast and human Mediator complexes and have a better understanding of their interactions with the Pol II pre-initiation complex (PIC). Herein, we provide a summary of recent achievements and discuss their implications for future studies of Mediator and its role in gene regulation.

The repetitive heptads in the C-terminal domain (CTD) of RPB1, the largest subunit of RNA Polymerase II (Pol II), play a critical role in the regulation of Pol II-based transcription. Recent findings on the structure of the CTD in the pre-initiation complex determined by cryo-EM and the novel phase separation properties of key transcription components offers an expanded mechanistic interpretation of the spatiotemporal distribution of Pol II during transcription. Current experimental evidence further suggests an exquisite balance between CTD’s local structure and an array of multivalent interactions that drive phase separation of Pol II and thus shape its transcriptional activity.

Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression.

Splicing of precursor mRNAs is a hallmark of eukaryotic cells, performed by a huge macromolecular machine, the spliceosome. Four DEAH-box ATPases are essential components of the spliceosome, which play an important role in the spliceosome activation, the splicing reaction, the release of the spliced mRNA and intron lariat, and the disassembly of the spliceosome. An integrative approach comprising X-ray crystallography, single particle cryo electron microscopy, single molecule FRET, and molecular dynamics simulations provided deep insights into the structure, dynamics and function of the spliceosomal DEAH-box ATPases.

DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1–RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1–RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.