Initiation at AUGUG and GUGUG sequences can lead to translation of overlapping reading frames in E. coli.

During initiation, the ribosome is tasked to efficiently recognize open reading frames (ORFs) for accurate and fast translation of mRNAs. A critical step is start codon recognition, which is modulated by initiation factors, mRNA structure, a Shine Dalgarno (SD) sequence and the start codon itself. Within the Escherichia coli genome, we identified more than 50 annotated initiation sites harboring AUGUG or GUGUG sequence motifs that provide two canonical start codons, AUG and GUG, in immediate proximity. As these sites may challenge start codon recognition, we studied if and how the ribosome is accurately guided to the designated ORF, with a special focus on the SD sequence as well as adenine at the fourth coding sequence position (A4). By in vitro and in vivo experiments, we characterized key requirements for unambiguous start codon recognition, but also discovered initiation sites that lead to the translation of both overlapping reading frames. Our findings corroborate the existence of an ambiguous translation initiation mechanism, implicating a multitude of so far unrecognized ORFs and translation products in bacteria.

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. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

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. We introduced m(5)C, m(6)A, Ψ or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs.

Atomic mutagenesis at the ribosomal decoding site.

Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an in vitro reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding.

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation.

Following ribosomal peptide bond formation, the reaction products, peptidyl-tRNA and deacylated tRNA, need to be translocated from the A- and P-sites to the P- and E-sites, respectively. This process is facilitated by the GTPase elongation factor G (EF-G). The mechanism describing how the ribosome activates GTP hydrolysis is poorly understood in molecular terms. By using an 'atomic mutagenesis' approach, which allows the manipulation of specific functional groups on 23S rRNA nucleotides in the context of the entire ribosome, we disclose the adenine exocyclic N6 amino group at A2660 of the sarcin-ricin loop as a key determinant for triggering GTP hydrolysis on EF-G. We show that the purine pi system-expanding characteristics of the exocyclic functional group at the C6 position of A2660 are essential. We propose that stacking interactions of A2660 with EF-G may act as a molecular trigger to induce repositioning of suspected functional amino acids in EF-G that in turn promote GTP hydrolysis.

The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center.

Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.

Ribosome-associated GTPases: the role of RNA for GTPase activation.

The GTPase super-family comprises a variety of G proteins found in all three domains of life. Although they are participating in completely different processes like signal transduction, protein biosynthesis and regulation of cell proliferation, they all share a highly conserved G domain and use a common mechanism for GTP hydrolysis. Exact timing in hydrolyzing the bound GTP serves as a molecular switch to initiate diverse cellular reactions. Classical GTPases depend on external proteins to fire GTP hydrolysis (GAPs), and following the GTPase reaction to exchange GDP for GTP (GEFs), converting the GTPase into the active state again. In recent years it became clear that there are many GTPases that do not follow this classical switch mode scheme. Certain ribosome-associated GTPases are not reliant on other GEF proteins to exchange GDP for GTP. Furthermore many of these G proteins are not activated by external GAPs, but by evolutionarily ancient molecules, namely by RNA.