Lab Week 3

Evolution of the two components of telomerase ribonucleoprotein

The analysis for Telomerase reverse transcriptase (TERT) should be done at the amino acid level. Compared to Telomerase RNA Component (TERC), whose role is to serve as a RNA template for telomere synthesis without any function at the amino acid level, TERT will be translated into amino acids and it performs a highly conserved function in the polymerization mechanism across species. On the nucleic acid level, neutral mutations, such as synonymous base changes or base changes in the non-coding regions that do not affect the protein function, will not be vey informative in regards to evolution of TERT. Due to the function constrains of TERT, analysis of amino acids changes will shed more light on the divergence of this component across species.

It is necessary to adjust the alignment parameters according to the properties of the molecule being studied to get optimal results. One important distinction in alignment parameters I made between TERT and TERC is gap penalties. I used a higher gap-opening penalty for TERT – 14 – than TERC – 10. First, TERT is expected to be more conserved than TERC because of its function constrains. A higher gap penalty will allow for fewer gaps and the resulting identities are more likely to be the real conserved region rather than from random pairing. Second, TERT is studied at the amino acid level. In general, proteins are less tolerant to having one or several residues chopped away or inserted than a residual replacement in a position. Therefore, Gaps and insertions at the amino acid level should be more rare than those at the DNA level. A lower gap penalty is set for TERC because it would allow the detection of conserved region even with frequent deletions/insertions.

The Telomerase RNA Component (TERC) evolves much faster than Telomerase reverse transcriptase (TERT). The total alignment score for TERC is 65969 and that for TERT is 107134, which is almost two folds higher, indicating that TERT has more conserved regions than TERC does. TERC diverges more than TERT does across the species. This is in agreement with the hypothesis. Without function constrains, TERC is expected to a have a higher proportion of neutral mutation sites so that a significant fraction of the base substitutions can be fixed. On the other hand, a larger proportion of the amino acid replacements in TERT are probably very deleterious because it may change the central function of this protein and thus is eliminated. Therefore, TERT evolves slower than TERC.

TERT has two functional domains - one for RNA binding and the other for the reverse transcriptase activity. Because these two domains have crucial functions, they tend to be conserved across species. From the alignments, the highly conserved domains are detected by eyeballing (finding the consecutive asterisks). The first conserved region using the human homolog as a reference is from bp1539 to bp 2181 and the second one from bp 2499 to bp 2850. The two conserved regions are separated by a unconserved region. For TERC, the conserved region is from bp223 to bp 334. To be noted, the results are susceptible to human error and will vary among different operators.

Based on the molecular clock theory, the rate of evolution for a given protein is constant over evolutionary time and across lineages. To address this point in TERT, one need to plot the number of amino acid changes among species against the number of years since divergence. A linear regression will be performed. If the least-square line fitted is statistically significant, it proves that TERT evolves in clock-like fashion. This principle of molecular evolution also applies to DNA sequences, but the result should be interpreted with more caution, which is the case for TERC – a RNA template not translated. While proteins contain a total of twenty amino acids, DNA consists of only four nucleotides. This indicates that independent but identical mutations at nucleotides sites are much more likely than at amino acids level. What is more, a nucleotide site that undergoes two sequential mutations is more likely to return to its original state than amino acid and thus will not be detected by alignment. All the limitations will obscure the linearity of this relationship in TERC, making it difficult to determine whether TERC evolves in a clock-like fashion.

According to the BLAST results of TERT, it seems that there are no paralogs exist in the human genome. This could be explained by the fact that after the duplication event, all the paralogs undergo mutations that are deleterious and thus are eliminated in human genome. It can be hard to spot paralogs of TERC in the human genome. TERC is expected to have a high mutation rate itself. The redundant copy of duplication of this gene will have more freedom to evolve along any path while not being eliminated because it does not have central functionality.

Reference:
Telomerase. Database. EMBL-EBI (European Bioinformatics Institute).