Evidence of the involvement of E. coli 23S rRNA in decoding of termination codons was first provided by the isolation and characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. This dissertation describes the isolation and characterization of a group of nonsense suppressor mutations in E. coli 23S rRNA. In this work, one hypothesis was tested and several related questions were addressed.
The hypothesis was that, in addition to the first isolate, more 23S rRNA suppressors of specific nonsense mutations can be isolated. Along with this hypothesis were two questions: Are the nucleotides altered in 23S rRNA suppressors randomly distributed or located in specific regions? What are the codon specificities of the suppressors? To test the hypothesis, I screened for mutations in specific regions of the 23S rRNA gene targeted by PCR random mutagenesis that caused suppression of termination codons. Mutations located in a very short sequence known as the GTPase center were found and they all caused suppression of UGA but not UAG or UAA under same conditions. These results proved the hypothesis in general and specifically demonstrated that a group of nucleotides in the GTPase center of 23S rRNA are involved in decoding of the UGA codon.
I examined the in vivo function of L11, a ribosomal protein known to bind to the GTPase center of 23S rRNA. I showed that limitation of cellular availability or synthesis of L11 resulted in altered decoding of termination codons, indicating that L11 is directly involved in termination.
I asked the question of what structural features of the GTPase center rRNA are important for normal termination. This question was addressed by making site-specific changes of nucleotides in the GTPase center. Mutational analysis showed that a) the A/G base pair closing the hexanucleotide hairpin loop was dispensable for normal termination, b) the “U-turn” structure in the nucleotide 1093 to 1098 hexaloop is critical for normal termination, and c) nucleotides A1095 and A1067, necessary for the binding of thiostrepton, an antibiotic known to inhibit termination, to ribosomes, are also necessary for normal termination.
Involvement of a Region of 23S Ribosomal RNA of E. coli in Decoding of Termination Codons