tRNA imbalance promotes -1 frameshifting via near-cognate decoding

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Transfer RNA (tRNA) molecules are crucial for protein synthesis, translating genetic code from mRNA to amino acids.
• Specific tRNAs recognize and bind to particular mRNA codons.
• Codon-anticodon interactions are typically highly specific, ensuring accurate protein synthesis.

Purpose of the Study:

• To investigate the role of Escherichia coli glycine tRNA 1 (tRNAGly1) in frameshifting mutations.
• To understand the mechanism by which tRNAGly1 influences codon decoding, particularly near-cognate codons.
• To explore the potential for engineering tRNA properties for altered decoding.

Main Methods:

• Genetic selection for multicopy suppressors of a frameshift mutation.
• Analysis of the suppression spectrum of the identified multicopy suppressor.
• Peptide sequencing of the suppressed protein product.
• Introduction of a specific tRNA modification (cytosine at position 32) from Mycoplasma mycoides into E. coli tRNAGly1.

Main Results:

• Increased levels of wild-type tRNAGly1 were found to cause -1 frameshifting.
• tRNAGly1 was shown to promote the decoding of near-cognate GGA codons, in addition to its cognate GGG codons.
• This near-cognate decoding occurs via a two-out-of-three reading mechanism.
• Introducing a Mycoplasma mycoides tRNA feature (C32) into E. coli tRNAGly1 enhanced doublet decoding efficiency.

Conclusions:

• The concentration of GGG-specific tRNAGly1 relative to GGA-decoding tRNAGly2 influences the reading of GGA codons.
• tRNAGly1 can mediate frameshifting through near-cognate decoding of GGA codons.
• Modifications in tRNA structure, like the C32 modification, can enhance non-discriminatory codon decoding.