A study published in Science (2009) investigates the factors influencing gene expression in Escherichia coli. Researchers engineered a synthetic library of 154 genes, all encoding the same green fluorescent protein (GFP), with random synonymous mutations. When expressed in E. coli, GFP protein levels varied 250-fold. mRNA levels, degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Instead, mRNA folding near the ribosomal binding site explained over half the variation in protein levels. The study found that mRNA folding and translation initiation rates play a major role in shaping gene expression, while codon bias influences global translation efficiency and cellular fitness. The researchers analyzed the effects of synonymous mutations on gene expression and found that mRNA structure, particularly near the ribosomal binding site, was a key determinant of protein levels. They also found that mRNA folding energy near the start codon strongly correlated with fluorescence levels. This suggests that tightly folded mRNAs may obstruct translation initiation, reducing protein synthesis. The study also found that bacterial growth rates were influenced by codon bias, with higher codon adaptation (CAI) correlating with faster growth. The study highlights the importance of mRNA structure in determining gene expression and challenges the traditional view that codon bias is the main determinant of gene expression. The findings suggest that translation initiation, not elongation, is the rate-limiting step in gene expression. The study also shows that codon adaptation may be selected for to improve global translation efficiency, rather than individual gene expression. The results have implications for understanding how gene expression is regulated in bacteria and may inform the design of synthetic genes for biotechnology applications.A study published in Science (2009) investigates the factors influencing gene expression in Escherichia coli. Researchers engineered a synthetic library of 154 genes, all encoding the same green fluorescent protein (GFP), with random synonymous mutations. When expressed in E. coli, GFP protein levels varied 250-fold. mRNA levels, degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Instead, mRNA folding near the ribosomal binding site explained over half the variation in protein levels. The study found that mRNA folding and translation initiation rates play a major role in shaping gene expression, while codon bias influences global translation efficiency and cellular fitness. The researchers analyzed the effects of synonymous mutations on gene expression and found that mRNA structure, particularly near the ribosomal binding site, was a key determinant of protein levels. They also found that mRNA folding energy near the start codon strongly correlated with fluorescence levels. This suggests that tightly folded mRNAs may obstruct translation initiation, reducing protein synthesis. The study also found that bacterial growth rates were influenced by codon bias, with higher codon adaptation (CAI) correlating with faster growth. The study highlights the importance of mRNA structure in determining gene expression and challenges the traditional view that codon bias is the main determinant of gene expression. The findings suggest that translation initiation, not elongation, is the rate-limiting step in gene expression. The study also shows that codon adaptation may be selected for to improve global translation efficiency, rather than individual gene expression. The results have implications for understanding how gene expression is regulated in bacteria and may inform the design of synthetic genes for biotechnology applications.