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Evolutionary site rates were estimated with the maximum likelihood method implemented in the TREE-PUZZLE program v5.1 [ 41, 42].
For the estimation of the evolutionary site rates with TREE-PUZZLE, we have chosen a heterogeneity rate model that was compared with the null model, which assumes a uniform rate among sites.
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Previous computational work on elongation factors (EF) has nicely demonstrated that identifying evolutionary site-rate shifts coupled with analyses of three-dimensional structures of the protein family can pinpoint sites that are likely important in functional divergence and structural change between bacterial elongation factor Tu (EF-Tu) and eukaryotic elongation factor 1α (EF-1α) [ 24].
DB carried out the codon usage analysis, identified the co-evolutionary sites, participated in the identification of positively selected sites, helped to draft and revise the manuscript, and supervised the project.
It was shown that this evolutionary conserved site was vital in positively regulating expression of NDRG1 [ 65].
In four genes (petA, rpl14, RNApolB, rps18), the opposite was true: in most plant species, evolutionary conserved sites had lesser helix-forming propensity than other sites.
Given the premise that evolutionary conserved sites are more likely to be functionally relevant, we tested these conserved sites for their ability to bind TF in vivo.
In highly biased genes, major codons are more likely to be used at evolutionary conserved sites, while minor codons are more likely to be used at evolutionary variable sites.
The recent finding that elongation speed (inferred from mRNA structure) is slowed down at evolutionary conserved sites [ 36] would be in agreement with our results due to the known relationship between functional sites and sequence conservation.
In addition, four of these six heteroplasmic mutations affected evolutionary fast sites [40,41].
These evolutionary variable sites may conceivably be the acceptors for interaction with other regulator proteins.
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