Efficient expression of gene variants that harbour AGA codons next to the initiation codon

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The efficiency of gene expression is influenced by codon usage, particularly in early open reading frame (ORF) sequences.
• Arginine codons, specifically AGA and AGG, have shown varied effects on translation depending on their position within a gene.

Purpose of the Study:

• To investigate the impact of specific arginine codon pairs at the second and third positions of the open reading frame on lacZ gene expression.
• To understand the rules governing the influence of early ORF sequences on translation efficiency.

Main Methods:

• Analysis of lacZ gene variants with different pairs of arginine codons (AGA, AGG, CGN) at the second and third positions.
• Measurement of lacZ expression levels and corresponding mRNA levels.
• Investigation of peptidyl-tRNA accumulation and ribosome stalling in specific variants.

Main Results:

• Identical AGA or AGG codon pairs at early positions enhanced gene expression, similar to AAA codons.
• Identical CGN codon pairs at early positions resulted in very inefficient gene expression.
• Mixed combinations of AGA and CGN codons generally led to efficient lacZ expression.
• The lacZ AGA AGA variant in pth cells showed ribosome stalling, suggesting aminoacylated-tRNA depletion.

Conclusions:

• Early ORF sequences, particularly the presence of adenine in arginine codons (AGA/AGG), positively influence gene expression.
• The position of arginine codons is critical; early positions behave differently than internal positions regarding translation efficiency.
• Ribosome stalling can occur due to the depletion of specific aminoacylated-tRNAs, even with favorable early codon sequences.