RESUMO
Telomerase is an enzyme that catalyzes addition of telomeric repeat sequences to the 3'-termini of eukaryotic chromosome DNA. The catalytic core of telomerase consists of a protein component, telomerase reverse transcriptase (TERT), for the catalysis and an RNA component, telomerase RNA (TR), containing the template for the sequence. Human telomerase RNA (hTR) consists of 451 nucleotides (nt) and contains consecutive G-stretches in the 5'-terminal region. We examined the effects of the 5'-terminal sequence (nt 1-17) in hTR, which is assumed to be a single-stranded region (region 1), on interaction and telomerase activity in vitro. Mutation and binding experiments for hTR and its variants suggest that region 1 has repressive effects on telomerase activity by interaction with the region(s) in the 3'-half part. We prepared various hTR variants with mutations in region 1 and two possible target regions (region 2: nt 229-244; region 3: nt 284-297). Studies on these variants showed that region 1 can interact with regions 2 and 3 and the interactions between regions 1 and 3 may contribute to the repressive effects of region 1. We found that a mutation in region 2 markedly enhances telomerase activity. We also found that some deletion and sequence mutations in region 1 enhance the activity.
Assuntos
RNA/química , Telomerase/genética , Sequência de Bases , Dicroísmo Circular , DNA/química , DNA Antissenso/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Quadruplex G , Guanina/química , Humanos , Mutação , RNA/genética , Telomerase/metabolismoRESUMO
It is reported that a single stranded region at 5'-terminus of human telomerase RNA (hTR) affects telomerase activity, though the sequences of the region are not conserved among vertebrate species. The mechanism of this phenomenon is not known. We examined binding affinity of an RNA oligomer (R17), which corresponds to the single stranded region (1-17) at 5'-terminus of hTR, to deletion variants of hTR. R17 showed higher affinity toward the 3'-half part of hTR than that for the 5'-half part. We chose two regions of the 3'-half part, where R17 is assumed to bind, and prepared variants of hTR in the single stranded region and the selected regions. The interactions and telomerase activities of these variants were examined. We also prepared an RNA interaction domain of the protein component (hTERT) of telomerase and its variants and tried to elucidate influence on interactions with hTR.
Assuntos
RNA não Traduzido/química , Telomerase/química , Sequência de Bases , Sequência Conservada , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA , RNA Longo não Codificante , RNA não Traduzido/metabolismo , Telomerase/metabolismoRESUMO
Human telomerase RNA (hTR) is assumed to contain a 17-nt segment in a single-stranded state at the 5'-terminus. There are four stretches of consecutive G residues, which are apt to form a quadruplex structure, in the segment. It is reported that deletion of some part of this region enhances telomerase activity when the core telomerase enzyme is reconstituted by addition of telomerase reverse transcriptase. To elucidate the reason for such effects, we constructed hTR mutants with deletion of the segment (hTRdelta20) and with the segment containing four G-to-A displacement to interrupt the G-stretch sequences (hTR17A) and their activity was compared with that of the wild-type hTR (hTRW). hTRdelta20 showed much higher activity than the other while hTR17A showed activity similar to that of hTRW. This result suggests that the lower activity for full-length hTR is not due to putative quadruplex formation.
