Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum

When the K⁺ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.     

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA3 ₃ ^Leu ) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNA^Leu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNA^Leu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.     

The Mystery of Two Straight Lines in Bacterial Genome Statistics

In special coordinates (codon position-specific nucleotide frequencies), bacterial genomes form two straight lines in 9-dimensional space: one line for eubacterial genomes, another for archaeal genomes. All the 348 distinct bacterial genomes available in Genbank in April 2007, belong to these lines with high accuracy. The main challenge now is to explain the observed high accuracy. The new phenomenon of complementary symmetry for codon position-specific nucleotide frequencies is observed. The results of analysis of several codon usage models are presented. We demonstrate that the mean-field approximation, which is also known as context-free, or complete independence model, or Segre variety, can serve as a reasonable approximation to the real codon usage. The first two principal components of codon usage correlate strongly with genomic G+C content and the optimal growth temperature, respectively. The variation of codon usage along the third component is related to the curvature of the mean-field approximation. First three eigenvalues in codon usage PCA explain 59.1%, 7.8% and 4.7% of variation. The eubacterial and archaeal genomes codon usage is clearly distributed along two third order curves with genomic G+C content as a parameter.     

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca(2+) regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in Escherichia coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5'-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5' but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation.     

Variation in macronuclear genome content of three ciliates with extensive chromosomal fragmentation: a preliminary analysis

The genome architecture of ciliates, including features such as nuclear dualism and large-scale genome rearrangements, impacts gene and genome evolution in these organisms. To better understand the structure of macronuclear chromosomes in ciliates with extensively processed chromosomes, a sample of complete macronuclear chromosomes was sequenced from three ciliate species: Metopus es (Class [Cl]: Armophorea), Nyctotherus ovalis (Cl: Armophorea), and Chilodonella uncinata (Cl: Phyllopharyngea). By cloning whole macronuclear chromosomes into a plasmid vector, we generated nine clones from each of M. es and C. uncinata, and 37 clones from N. ovalis. Analysis of these macronuclear chromosomes provides insight into the evolution of genome features such as chromosome content, gene structure, and genetic code. Phylogenetic patterns can be found in telomere structure and codon usage, which are both more similar in M. es and N. ovalis than C. uncinata. In addition, we provide evidence of lateral transfer of a bacterial endo-beta-mannanase gene onto a M. es chromosome and report the discovery of a 42-bp conserved sequence motif within N. ovalis untranslated regions.     

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC

Abstract A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-β-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50 909 Da. The enzyme is functional and extremely overexpressed in E. coli .     

Optimality of codon usage in Escherichia coli due to load minimization

The canonical genetic code is known to be highly efficient in minimizing the effects of mistranslational errors and point mutations, an ability which in term is designated "load minimization". One parameter involved in calculating the load minimizing property of the genetic code is codon usage. In most bacteria, synonymous codons are not used with equal frequencies. Different factors have been proposed to contribute to codon usage preference. It has been shown that the codon preference is correlated with the composition of the tRNA pool. Selection for translational efficiency and translational accuracy both result in such a correlation. In this work, it is shown that codon usage bias in Escherichia coli works so as to minimize the consequences of translational errors, i.e. optimized for load minimization.