Abstract
The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.
Highlights
Protein sequence is shaped by many evolutionary constraints, acting at different levels of the gene expression process
C-terminal amino acid and codon composition in bacteria is biased We investigated biases in codon and amino acid composition of the C-terminal region of bacterial protein sequences
When studying all species in the bacterial kingdom, we found that the amino acid composition at the last position upstream of the stop codon differed significantly from the bulk amino acid composition (Fig 1A)
Summary
Protein sequence is shaped by many evolutionary constraints, acting at different levels of the gene expression process. Identifying which sequence features determine the efficiency and accuracy of protein expression has been the subject of intense research. Studies showed a differential preference for specific codons upstream of the stop codon in Escherichia coli (Brown et al, 1990; Arkov et al, 1993; Bjornsson et al, 1996; Berezovsky et al, 1999) and Bacillus subtilis (Rocha et al, 1999; Palenchar, 2008) proteins. The properties of the last two amino acids were shown to modulate the efficiency of translation termination at the UGA stop codon context in E. coli (Bjornsson et al, 1996), in particular for highly expressed genes. Changes in the efficiency of translation termination and recognition of the C-terminal region by the protein degradation machinery are two potential mechanisms that could drive preferences in the C-terminal composition of proteins
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