Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step.

Eukaryotic Translation Elongation is Modulated by Single Natural Nucleotide Derivatives in the Coding Sequences of mRNAs.

RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs.

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N of purines and the N of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition.

Methylated mRNA Nucleotides as Regulators for Ribosomal Translation.

Methylated RNA nucleotides were recently discovered to be highly abundant in RNAs. The effects of these methylations were mainly attributed to altered mRNA stabilities, protein-binding affinities, or RNA structures. The direct impact of RNA modifications on the performance of the ribosome has not been investigated so far.

mRNA modifications: Dynamic regulators of gene expression?

The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), pseudouridine (Ψ) and N(1)-methyladenosine (m(1)A) have been found within open reading frames of mRNAs.

Translating the epitranscriptome.

RNA modifications are indispensable for the translation machinery to provide accurate and efficient protein synthesis. Whereas the importance of transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications has been well described and is unquestioned for decades, the significance of internal messenger RNA (mRNA) modifications has only recently been revealed. Novel experimental methods have enabled the identification of thousands of modified sites within the untranslated and translated regions of mRNAs.

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation.