Discussion 5

P-loop NTPases are the most abundant protein in cellular organisms, constituting 10-18% of all gene products. They are distinguished by the Walker A motif (consensus GxxxxGK[ST]), Walker B motif (consensus hhhDxxG, where h = hydrophobic residue), and the [NT]KxD motif which is unique to P-loop NTPases. The Walker B and [NT]KxD motifs indicate specificity towards GTP (Leipe, D. et. al. 2002). MSA of known and suspected GTPases displayed high conservation of these characterising motifs indicating that 1pujA is a GTPase. Furthermore, all sequences aligned had the [NT]KxD motif N-terminal of the Walker A motif, indicative that they are all part of YlqF family of P-loop NTPase superfamily (Leipe, D. et. al. 2002). It has previously been hypothesised that the Last Universal Common Ancestor (LUCA) to all extant life forms possessed several GTPases. If a particular GTPase family is widely represented in the three primary kingdoms (Archaea, Bacteria, and Eukaryota), this is evidence for presence in LUCA. This is supported if phylogenetic tree conforms to the “standard model” topology, having bacterial and archeo-eukaryotic primary clades. Conversely, a different topology such as a bacteria-eukaryote grouping could indicate presence in LUCA but ancestral form in eukaryotes displaced by horizontal gene transfer (HGT) (Leipe, D. et. al. 2002). 1PUJA was found to be widely represented in the three primary kingdoms and conformed to the “standard model” topology. All of which is suggestive of the presence in LUCA. May have been transferred from bacteria to eukaryotes once during the early stages of eukaryotic evolution (probably from the proto-mitochondrion), and a second time from chloroplasts to plants. It has been stipulated that GTPase activity is a result of adaptation. GTP is more constant within a cell and not subject to the same fluctuations as ATP. Hence specificity for use of GTP as a substrate recruited in crucial functions such as translation (Leipe, D. et. al. 2002).

Structural analysis shows that YlqF is …

A functional analysis of 1pujA indicated that our protein is a GTPase.

ProFunc returned a number of promising results including Interpro, PDB, SSM and DALI and 3D functional template searches. These results while not being significant did support our hypothesis. The result of the UniProt search however was significant. A multiple sequence alignment search returned 10 significant matches 2 of which had a percentage identity higher then 80%. Both of these sequences were listed as YlqF proteins. All of the proteins featured in these results were either GTPases or GTP-binding proteins. Therefore this sequence search supports our hypothesis of 1pujA being a GTPase.

The SymAtlas results show the expression levels of ortologs for 1pujA in different tissues of both mice and humans. The results indicated that these proteins are widely distributed in all tissues of both animals. This is consistent with a protein essential for fundamental biological processes. Though the results do not provide a greater level of detail than this, a GTPase would fall into this category, further supporting our hypothesis.

Structural, functional and evolutionary analyses collectively suggest that YlqF is a GTPase. High conservation of YlqF over large spans of evolutionary time (Figure ?) indicate strong stabilising selection and suggest a role in one or several crucial biological roles (Leipe, D. et. al. 2002). Functional results indicate YlqF has a very important function inferred from the results of expression in all tissues of humans and mice. In conclusion, YlqF is a GTPase likely to be involved in a fundamental biological process such as translation. In support of the study by Matsuo et. al. (2002) in which it was hypothesised that YlaF is needed for correct assembly of the 50S ribosomal subunit.