Codon Clusters Research Articles

Natural tuning of restriction endonuclease synthesis by cluster of rare arginine codons

Restriction–modification (R-M) systems are highly widespread among bacteria and archaea, and they appear to play a pivotal role in modulating horizontal gene transfer, as well as in protecting the host organism against viruses and other invasive DNA particles. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). If the cell is to survive, the counteracting activities as toxin and antitoxin, must be finely balanced in vivo. The molecular basis of this regulatory process remains unclear and current searches for regulatory elements in R-M modules are focused mainly at the transcription step. In this report, we show new aspects of REase control that are linked to translation. We used the EcoVIII R-M system as a model. Both, the REase and MTase genes for this R-M system contain an unusually high number of rare arginine codons (AGA and AGG) when compared to the rest of the E. coli K-12 genome. Clusters of these codons near the N-terminus of the REase greatly affect the translational efficiency. Changing these to higher frequency codons for E. coli (CGC) improves the REase synthesis, making the R-M system more potent to defend its host against bacteriophages. However, this improved efficiency in synthesis reduces host fitness due to increased autorestriction. We hypothesize that expression of the endonuclease gene can be modulated depending on the host genetic context and we propose a novel post-transcriptional mode of R–M system regulation that alleviates the potential lethal action of the restriction enzyme.

Gene Expression Prediction and Hierarchical Clustering Analysis of Plant CCD genes

Plant carotenoid cleavage dioxygenase (CCD) catalyses the formation of industrially important apocarotenoids. Here, we applied codon-based classification for 72 CCD genes from 35 plant species using hierarchical clustering analysis. The codon adaptation index (CAI) and relative codon bias (RCB) were utilized to estimate the level of gene expression. The codon-based cluster tree result shows neatly clustered subclass of CCD genes except BoCCD1 gene of Bixa orellana. Correlation analysis of CAI values with RCB indicates an overall low-level expression of CCD across different species. Similarly, the closeness in the codon cluster with same CAI values was not reflected in 3-D structural report of selected CCD genes. These finding not only enhances our insights into the classification of CCD gene across the species but also identifies the critical factors responsible for this variation, which could aid in prediction of gene expression and function for newly reported CCD genes.

Detecting slow-translating regions in E.coli.

We present an application of the spatial scan statistic to identifying clusters of slow-translating codons in E.coli. We use an estimate of the time taken to process each codon based on the availability of transfer RNA. We analyse our findings using experimental measurements, and provide support for a well-known hypothesis related to the process of protein production.

Clustering of Codons with Rare Cognate tRNAs in Human Genes Suggests an Extra Level of Expression Regulation

In species with large effective population sizes, highly expressed genes tend to be encoded by codons with highly abundant cognate tRNAs to maximize translation rate. However, there has been little evidence for a similar bias of synonymous codons in highly expressed human genes. Here, we ask instead whether there is evidence for the selection for codons associated with low abundance tRNAs. Rather than averaging the codon usage of complete genes, we scan the genes for windows with deviating codon usage. We show that there is a significant over representation of human genes that contain clusters of codons with low abundance cognate tRNAs. We name these regions, which on average have a 50% reduction in the amount of cognate tRNA available compared to the remainder of the gene, RTS (rare tRNA score) clusters. We observed a significant reduction in the substitution rate between the human RTS clusters and their orthologous chimp sequence, when compared to non–RTS cluster sequences. Overall, the genes with an RTS cluster have higher tissue specificity than the non–RTS cluster genes. Furthermore, these genes are functionally enriched for transcription regulation. As genes that regulate transcription in lower eukaryotes are known to be involved in translation on demand, this suggests that the mechanism of translation level expression regulation also exists within the human genome.

Distribution of Rare Triplets Along mRNA and Their Relation to Protein Folding

It is believed that pausing during mRNA translation plays some role in ensuring proper folding of newly synthesized sections of a protein chain. Such pausing occurs when rare triplets are encountered in the mRNA, as it takes additional time for the corresponding rare species of tRNA to be delivered. To determine whether pause sites are non-randomly distributed along prokaryotic mRNA (cDNA), we have located clusters of rare triplets in cDNA sequences from 21 different bacteria. From the individual profiles of local codon frequencies calculated with various windows, the positions of the clusters of the rarest codons were taken for generation of the combined histograms of positional preferences of the pause sites. The histograms show that in the prokaryotic sequences, the pause sites are located preferentially at the start positions and at about 155 triplets from the starts. To verify the generality of these observations, the data are grouped in six independent sets about 500 sequences each, all revealing the same features. A less prominent maximum is also seen at the triplet position 75. Judging by the amplitude of the peak at 155 triplets, an optimal cluster size is estimated to equal 18 triplets. The distance 155 closely corresponds to the sizes of typical protein folds and to earlier estimated prokaryotic protein sequence segments. This supports the suggestion of a role for translation pausing in the cotranslational folding of protein domains. The profiles of rare codons in mRNA can serve in the detection or prediction of boundaries between protein domains.

Overexpression and purification of the hepatitis B e antigen precursor

Circumstantial evidence suggests that the secreted hepatitis B virus (HBV) e antigen (HBeAg) and/or its 22 kDa precursor (P22) have an essential role in the establishment of persistent infection. In order to identify cellular proteins that could interact with P22, large amounts of this protein are required to perform pull-down assays. A plasmid was constructed encoding a recombinant P22 with a Histidine-tag at its N-terminal extremity (P22r). The initial attempts to overexpress P22r in a conventional Escherichia coli strain failed, most likely due to the presence of rare AGA/AGG codon clusters in the 3′ part of the gene. To overcome this difficulty, P22r was overexpressed in the Epicurian coli BL21- codonplus™ ( DE3)- RIL strain, which possesses extra copies of the ArgU gene that encodes the tRNA AGA/AGG. In this strain, P22r was overexpressed successfully and then purified in milligram quantities by metal affinity chromatography on Ni 2+-chelated His-Bind resin. The purified recombinant protein P22r was able to interact with a cellular protein (P32), which had previously been shown to co-immunoprecipitate with native P22, indicating that at least some of the P22r molecules were folded correctly.

Genetic code and optimal resistance to the effects of mutations.

This paper deals with the notion of resistance of the genetic code to the effects of mutations. We measure the resistance of a group of t codons as the number of pairs of those which differ from each other in only one of their three bases. We find for each value of t the maximum possible value of the resistance and we describe some groups of codons giving this value. Important examples of such configurations are found in the genetic code, among these are the groups of synonymous codons, as observed elsewhere, and the cluster of codons which have an hydrophobic amino acid for translation.