In this study, the charge distribution of all E. coli
sec dependent signal peptides was analysed. This revealed a massive bias for lysine at P2, which could be attributed to a bias for the lysine codon AAA. The preference for lysine codon AAA at P2 was experimentally shown not to be a requirement for a positive charge. Two studies have shown that AAA at P2 is the best initiator of translation (16
). We propose that the selective pressure at P2 is for codons that promote faster translation initiation efficiencies. There does not appear to be any selective pressure at P2 for residues that do not promote N-terminal methionine removal.
Sequences rich in adenine nucleotides immediately downstream of the start codon have been shown to enhance gene expression, presumably by enhancing translation initiation (22
). As lysine is encoded by AAA and AAG, it could be these factors that enhance choice of lysine at P2 and P3, not a requirement for a positive charge. This is supported by the ratio of lysine to arginine, which is 4.05:1 at P2 and drops at every position down to 0.41:1 by P9. If the requirement were simply for a positive charge, then one would not expect preferential usage of one basic amino acid over another. This enhances the idea that secretory proteins require higher translation initiation rates, but raises the question why that is necessary?
Signal peptides also contain the highest levels of non-optimal codons seen anywhere in the genome (23
). Studies have shown that the insertion of consecutive non-optimal codons downstream of the start codon significantly lowers protein production compared to insertion of the same codons further downstream (24–26
), due to ribosomes dissociating from the transcript prematurely (27
). Hence for secretory proteins it is likely that ribosomes would disassociate prematurely due to the high levels of non-optimal codons in the signal sequence. Preferential use of AAA at P2, and the high use of adenine rich nucleotides at P3, which promotes rapid translation initiation, would help to counteract that effect, as subsequent ribosomes would quickly replace the previously dissociated ones. This would likely result in more ribosomes per transcript. Conversely factors that promote slow translation initiation would likely result in the spacing between ribosomes being greater, as one ribosome would be able to translate more codons before the next one commences translation.
Biasing codons to ensure rapid translation initiation could help recycle chaperones required for export. For example the molecular chaperone SecB delivers the presecretory protein to SecA, while SRP delivers the presecretory to FtsY (1
). Both SecA and FtsY are inner membrane proteins. Once directed to these proteins, the chaperone is free to associate with a new nascent peptide. If ribosomes are close together on an mRNA transcript, due to increased translation initiation efficiencies, this could allow efficient binding to a new nascent peptide emerging from an upstream ribosome. This time factor may be important, as proteins must be in a loosely folded state to allow protein export (28
). If it takes longer for the chaperone to find the next nascent peptide, this could mean the nascent peptide folds into a conformation incapable of export.
This study, as well as others (6–9
), has found that a positive charge is not required for protein export. Studies have found that a net negative charge is deleterious for export, resulting in increased amounts of unprocessed precursor (7–9
). Other than the extreme bias for lysine at P2 and P3, which could be to promote high translation initiation frequencies, there was no bias for a positively charged residue at any other position. This raises the possibility that the overall selective force in the entire N-region of signal peptides is to avoid a net-negative charge. Supporting this is the fact that the negatively charged residues glutamic acid and aspartic acid occur on average 0.01 times from P2-P10 in secretory proteins, compared to 0.81 times for all other genes (data not shown). Given that most secretory proteins are exported to the membrane by SRP or SecB (30
), there is no requirement for the positive charge to interact with the membrane to initiate export. Once at the membrane, a net-negative charge would interfere with insertion into the membrane, due to the negatively charged phospholipids. Hence the observation that sec dependent signal peptides contain a positive charge at the N-region could be due to selection for lysine at P2 and P3 to promote high translation initiation efficiencies, and an overall selection against a net-negative charge.