A single mutation in 16S rRNA that affects mRNA binding and translation-termination

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• A single base change in 16S ribosomal RNA (rRNA) from cytosine to guanine at position 726 (C726G) in Escherichia coli (E. coli) has been previously linked to significant alterations in protein synthesis.
• Understanding the precise molecular mechanisms underlying these changes is crucial for comprehending bacterial gene expression regulation.

Purpose of the Study:

• To investigate the specific effects of the 16S rRNA C726G mutation on messenger RNA (mRNA) binding to the ribosome.
• To elucidate the impact of this mutation on the process of translation termination, particularly concerning nonsense codon suppression.

Main Methods:

• Utilized the in vitro toeprinting technique to assess the binding affinity of mutant (726G) and wild-type 30S ribosomal subunits to mRNA.
• Performed in vivo expression studies of mutant ribosomes to observe their effect on nonsense codon suppression.
• Conducted in vitro experiments to evaluate the binding affinity of mutant ribosomes to Release Factor-2 (RF-2).

Main Results:

• The toeprinting assay demonstrated that 30S ribosomal subunits harboring the 726G mutation exhibit altered mRNA binding affinity compared to wild-type subunits.
• In vivo expression of the mutant ribosomes led to the exclusive suppression of the UGA nonsense codon.
• In vitro studies confirmed that the mutant ribosomes possess an altered binding affinity for Release Factor-2, correlating with the observed nonsense codon suppression.

Conclusions:

• The 16S rRNA C726G mutation significantly impacts both mRNA binding and translation termination in E. coli.
• Altered ribosome interaction with mRNA and Release Factor-2 is the underlying mechanism for the observed UGA nonsense codon suppression.
• This mutation provides a valuable tool for studying the intricate processes of translation initiation, elongation, and termination in bacteria.