Manipulating the genetic code for membrane protein production: what have we learnt so far?

Abstract

With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50 years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Fig. 1. The genetic code and its relation to hydrophobicity of the encoded amino acids

(A) Typical schematic representation of the genetic code, illustrating how the four different nucleotides (U, C, A and G) encode 20 different amino acids and stop codons. Notably, hydrophobic amino acids such as phenylalanine (Phe), leucine (Leu), isoleucine (Ile), methionine (Met) and valine (Val) that are over-represented in transmembrane protein segments, all contain a uridine nucleotide in the second codon position. Codons with a U in the first position also tend to encode amino acids that are over-represented in membrane proteins. In the figure, amino acids that frequently occur in transmembrane segments have been emphasized with a lipid bilayer in the background. (B) Hydrophocity of the 20 different amino acids on a biological scale, specified as the free energy of membrane insertion (kcal/mol) when the indicated amino acid is placed in the middle of a 19-residue hydrophobic stretch [29]. Below is shown the codons that encode for the different amino acids, illustrating the U-bias of hydrophobic versus hydrophilic residues.

Fig. 2. Patterns of rare codon clusters are similar in membrane and soluble protein mRNAs

Rare codon clusters were identified in protein coding genes from the E. coli strain MG1655, using the method of Zhang et al. [25]. Membrane proteins (i.e. proteins containing at least one predicted transmembrane helix) were separated from soluble proteins using SCAMPI [68]. Transmembrane helices in the first 40 residues were excluded since these may constitute a signal peptide. (A) Rare codon clusters are more prevalent at the 5′ end. For each position in the sequence the fraction of residues present in a rare codon region was calculated. Running averages were calculated with a window size of 21 amino acids. (B) The fraction of proteins with rare codon clusters versus protein length (in amino acids). The solid lines represent transmembrane proteins (TM) and the dotted lines represent the soluble (nonTM) proteins. The red lines represent rare codons at or downstream of the 100^th amino acid whilst black lines represent rare codons within the first 100 amino acids.

Fig. 3. A model for how a rare codon cluster could pause translation and thereby affect the biogenesis of a membrane protein

The spacing of rare codon clusters and transmembrane helices in S. cerevisiae membrane proteins suggests that a pause might occur as a transmembrane helix exits the ribosome (see text for details). This event could influence (A) how efficiently the transmembrane helix partitions into the surrounding membrane, (B) how efficiently the transmembrane helix interacts with more N-terminally located transmembrane helices, or (C) how efficiently the regions flanking the transmembrane helix are glycosylated. The image was generated from a cryo-EM structure of the eukaryotic ribosome with bound Sec61 [69]. Figure courtesy of Dr. Shashi Bhushan, Wuerzburg University.