Studies on Translation Initiation and trans-Translation

Sammanfattning:  Translation initiation factor 1 (IF1) is an essential bacterial protein, without any clearly defined function in translation initiation. Same point mutants in this protein exhibit cold-sensitivity. In addition, a sequence specific increase in test protein expression has been observed for these mutants. Here in Paper I we analyze the parts of mRNA translation initiation region (TIR) involved in this effect, showing that Shine-Dalgarno sequence (SD), upstream enhancer region as well as linker between the initiation codon and SD are important. The similarity in action between the mutated IF1 and the antibiotic kasugamycin leads us to the suggestion that it occurs during the recently discovered proof-reading stage after the subunit joining step in the translation initiation. Deletion of yggJ and cspA genes involved in mRNA translation partially suppresses the cold-sensitivity phenotype of the R40D IF1 mutant. Deletion of the bipA gene confers even higher suppression confirming the previously assigned function of this protein in initiation of translation. In Paper II of this thesis proteome analysis of the IF1 mutation and kasugamycin action reveals some interesting features, such as increase in S6 polyglutamylation in the IF1 mutant cells or high level of GroEL-GroES protein in the cells treated with kasugamycin. A subset of genes having a similar TIR-specific increase in the expression under both conditions confirms previously made observations. Finally, in the Paper III we use cryo-electron tomography as well as chemical probing and computational modeling to address the question, how tmRNA moves on the ribosome during trans-translation. We observe that the tmRNA pseudoknots remain intact, while the mRNA part moves in the mRNA channel, allowing translation elongation to proceed.