Modeling Flexible Protein Structure With AlphaFold2 and Crosslinking Mass Spectrometry.

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2 and conformer selection using XL-MS data. For conformer selection, we developed two scores-the monolink probability score (MP) and the crosslink probability score (XLP)-both of which are based on residue depth from the protein surface.

Crystal structure of the translation recovery factor Trf from Sulfolobus solfataricus.

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNA to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S.

The SmAP2 RNA binding motif in the 3'UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro.

The SmAP1/2 proteins of the crenarchaeon Sulfolobus solfataricus interact with the exosome and stimulate A-rich tailing of transcripts.

The conserved Sm and Sm-like proteins are involved in different aspects of RNA metabolism. Here, we explored the interactome of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso) to shed light on their physiological function(s). Both, SmAP1 and SmAP2 co-purified with several proteins involved in RNA-processing/modification, translation and protein turnover as well as with components of the exosome involved in 3΄ to 5΄ degradation of RNA.

Binding of the 5'-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus.

The heterotrimeric archaeal IF2 orthologue of eukaryotic translation initiation factor 2 consists of the α-subunit, β-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs.

The Heptameric SmAP1 and SmAP2 Proteins of the Crenarchaeon Sulfolobus Solfataricus Bind to Common and Distinct RNA Targets.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive.

Back to translation: removal of aIF2 from the 5'-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus.

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5'-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5'-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs.

Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro.

Antisense regulation by transposon-derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon-derived paralogous RNAs, RNA-257(1-4), show extended complementarity to the 3' UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA-257(1) and increased 1183 mRNA levels.

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart.

X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly.

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.