Atomic mutagenesis of the small ribosomal subunit

The 30S ribosomal subunit is an important player in the process of protein synthesis. It is responsible for the correct binding of the mRNA and for a precise decoding process to ensure accurate protein synthesis. Additionally the 30S subunit has to undergo large conformational changes during translation. To decipher detailed aspects of decoding processes and the sterical prerequisites of translation we plan to apply an atomic mutagenesis approach. We will design and develop an in vitro reconstitution strategy for E. coli, which enables us to insert site specifically non- natural modifications at almost any position within the 16S rRNA. The basis for this approach is the use of fragmented 16S rRNA where a certain part of RNA is missing. The missing part will be compensated by a chemically synthesized oligonucleotide carrying the modification. This approach can be used to investigate 5`- or 3`- areas of the 16S rRNA or even intrinsic regions of the rRNA molecule. Depending on the research interest the reconstitution approach can be designed accordingly. This reconstitution strategy allows to literally dissect various aspects of translation in the small ribosomal subunit to so far unmet precision. Not only the nucleobases of 16S rRNA residues are amenable for modifications using this approach but also the ribose and the phosphate backbone, which is a significant advantage compared to so far applied biochemical experiments. One aspect we plan to investigate in greater detail is decoding. The interaction of the ribosome with the codon:anticodon helix provides the basis for the high accuracy of translation. Introducing modifications at nucleotides G530, A1492 and A1493, which are involved in decoding, might give us detailed insights into the contribution of single chemical groups on translation fidelity. Effects on the 30S structure, A-site binding, EF-Tu GTPase hydrolysis and in vitro translation will be investigated. This will add an additional layer of information to existing biochemical and structural data. An additional aspect of translation we want to concentrate on is tRNA translocation in the small ribosomal subunit. Crystal structures showed a steric block in the small ribosomal subunit, which impedes the translocation of the anticodon stem loops of tRNA from the P- to the E-site. This block is built by the nucleotides A790 and G1338 to U1341. Our reconstitution approach might allow us to elucidate if and how these nucleotides lock translocation. In summary, we want to establish a reconstitution approach which does not only enable the analysis of decoding and translocation to provide additional and important information on these processes but once set up this reconstitution strategy also allows the investigation of intersubunit bridges, communication between the subunits and antibiotic binding.

Protein biosynthesis is a central process in every living organism. More than 80 different proteins and RNA molecules have to be tightly orchestrated to allow an accurate and efficient process. The central player of translation is the ribosome. It is in charge of catalyzing peptide bond formations according to the sequence encoded by the mRNA. Whereas the peptidyl transferase center is located in the large ribosomal subunit, the decoding of the mRNA is performed at the small ribosomal subunit. During decoding the ribosome assures that only aminoacyl-tRNAs matching the codon of the mRNA are allowed to fully accommodate into the A-site subsequently followed by peptide bond formation, thereby prolonging the amino acid chain. According to the first high-resolution crystal structures the decoding is mainly carried out by nucleotides of the 16S rRNA, namely G530 and A1492 and A1493. They are supposed to sense the codon/anticodon interaction by forming hydrogen bonds with tRNA and mRNA nucleotides. A mismatch of this interaction leads to altered geometry of the tRNA/mRNA interaction and consequently the hydrogen bond network between tRNA, mRNA and the rRNA cannot fully be established. This leads to the subsequent rejection of the aa-tRNA. This model of decoding could explain earlier observation, but was mainly based on structural information. In this project we aimed to biochemically investigate the decoding process by performing atomic mutagenesis at the nucleotides A1492 and A1493. By altering the chemical composition of these two adenosines the hydrogen bond network can be specifically varied and their contribution to decoding investigated. To introduce these changes at positions 1492 and 1493, an in vitro reconstitution approach was employed. The ribosomes harboring a modified decoding site were then subjected to a variety of translation assays to investigate their ability to provide efficient and accurate protein synthesis.The results of our investigations indicated that this hydrogen bond network is not essential for an accurate decoding process. Single hydrogen bonds could be eliminated from the decoding site without significantly interfering with accurate translation of the mRNA. It appears that the decoding mechanism is more dependent on the geometry of the tRNA/mRNA interaction than on the hydrogen bonds that are formed. These findings are in line with and supported through recent structural studies and computational approaches.