Telomerase is an essential ribonucleoprotein enzyme for the stability of eukaryotic chromosome termini, which is recognized as telomere. Telomeres are specialized nucleoprotein complexes, consists of tandem DNA tracts (TTAGGG for all vertebrates) with specific telomeric protein bind to it. As the replication of eukaryotic chromosome can not performed all the way to the end of chromosome, telomere acts as a disposable buffer in replication. They are consumed during the replication of chromosome instead of the meaningful chromosome. Telomerase replenish the consuming of telomere by adding the specific DNA sequence to the termini. This process is regulated by the proteins bind to the telemetric DNA (like POT1 in humans). Besides of this, some other protein binds along (TRF1, TIN2, tankyrase in human) are part of the negative feedback of the elongation of the chromosome.
Telomerase is a reverse transcriptase as it carries its RNA template inside. However, it possesses an unusual character that its RNA template seems also acting in its enzyme function. The detail of the mechanism is still unclarified.
In this review, the structure, reaction and function of the telomerase will be discussed.
Telomerase ribonucleoprotein (RNP) was first purified from Euplotes aediculatus (1). The purification process gives out that telomerase complex had a molecular mass of about 230 kD, which RNA subunit counts for 66kD, and a pair of 43kD and 123kd of proteins counts for 166kD.(1) Through the experiment of photocross-linking, we could specify 123kD part binds to telomeric DNA part.
Through the SDS-polyacrylamide gels process, we could separates the polypeptides of the telomerase p123 subunits. Then place trypsin to digest telomerase p123, and further take nanoelectrospray tandem mass spectrometry to determine the amino acid sequences(2). Thus a miniatuized form of electrospray that allows mass spectrometric interrogation of minute analyte volumes.(3) After these process, 14 peptides of p123 would be sequenced:
Fig 1. Telomerase p123 subunit’s sequencing under tandem mass nanoelectrospray spectrometry. Part A is the unseparated peptide mixture under mass spectrum and part B is the tandem mass spectrum of the doubly charged precursor as the mass to charge ratio. (4)
The peptide sequences were assigned: T1, YLLFQR; T2, DYNEEDFQVL (VK,AR); T3, (L, N)WDVLMK; T4, SFLMNNLTHYFR; T5, TLYSWLQK; T6, ETLAEVQEK; T7, AMLGNELFR;T8, LQTSFPLSPSK; T9,(A,Q) TLFTNLFR; T10, (L,T) ALMPNLNLR; T11, LFAQTNLVATPR; T12, LESWMQVETSAK; T13, QYFFQDEWNQVR; T14, (E,D) SMNPENPNVNLLMR. (5)
Take 2 of these peptide sequences to design degenerate polymerase chain reaction (PCR) primers to amplify a part of p123’s gene, thus a gene library was prepared from it, then screen the fragment the integrate gene could be acquired. (4) It was found that p123 gene is 3279 base pair long, containing an continuous 1031-amino acid open reading frame.(4) The open reading frame predicts the protein is 122,562 daltons, matched the estimated size from SDS-polyacrylamide gel electrophoresis. The amino acids also matched well. These validate the experiment data.
Different species may bear the identical telomere sequence with each other but one species must have only one characteristic sequence. The repeating subunit of telomere sequence may be a perfect repeating one but may also an irregular one with variations on repeat unit. (6) But one thing must be unchanged, that one strand of DNA must contain clusters of G and another clusters of C. And the strand is rich of G is always at the 3’ end of the chromosome DNA strand. The telomerase synthesizes the G-rich strand DNA sequence. The elongation of telomere needs DNA primer to assist the telomerase so the telomeric repeats are added by polymerization in the 5’-3’ direction. Deoxynucleoside triphosphates (dNTPs) are...