Genes VII

Extracts of Tetrahymena contain an enzyme, called telomerase, which uses the 3′ VOH of the G+T telomeric strand as a primer for synthesis of tandem TTGGGG repeats. Only dGTP and dTTP are needed for the activity. The telomerase is a large ribonucleoprotein. It contains a short RNA component, 159 bases long in Tetrahymena, 192 bases long in Euplotes. Each RNA includes a sequence of 15 V22 bases that is identical to two repeats of the C-rich repeating sequence. This RNA provides the template for synthesizing the G-rich repeating sequence. Bases are added individually, in the correct sequence, as depicted in Figure 18.24. The enzyme progresses discontinuously: the template RNA is positioned on the DNA primer, several nucleotides are added to the primer, and then the enzyme translocates to begin again. The telomerase is a specialized example of a reverse transcriptase, an enzyme that synthesizes a DNA sequence using an RNA template (see 16 Retroviruses and retroposons).

The protein component has several subunits, one of which has the reverse transcriptase activity. The catalytic subunit (presumably) can act only upon the RNA template provided by the nucleic acid component (for review see Blackburn, 1991; Blackburn, 1992; Greider and Blackburn, 1987; Shippen-Lentz and Blackburn, 1990; Collins, 1999).

Telomerase synthesizes the individual repeats that are added to the chromosome ends, but does not itself control the number of repeats. Other proteins are involved in determining the length of the telomere. They can be identified by mutants in yeast that have altered telomere lengths. These proteins may bind telomerase, and thus influence the length of the telomere by controlling the access of telomerase to its substrate. Proteins that bind telomeres in mammalian cells have been found similarly, but less is known about their functions (for review see Zakian, 1995; Zakian, 1996).

What feature of the telomere is responsible for the stability of the chromosome end? Figure 18.25 shows that a loop of DNA forms at the telomere. The absence of any free end may be the crucial feature that stabilizes the end of the chromosome. The average length of the loop in animal cells is 5 V10 kb.

Figure 18.26 shows that the loop is formed when the 3′ single-stranded end of the telomere (TTAGGG)_n displaces the same sequence in an upstream region of the telomere. This converts the duplex region into a structure like a D-loop, where a series of TTAGGG repeats are displaced to form a single-stranded region, and the tail of the telomere is paired with the homologous strand (Griffith et al., 1999).

The reaction is catalyzed by the telomere-binding protein TRF2, which together with other proteins forms a complex that stabilizes the chromosome ends.

The minimum features required for existence as a chromosome are:

• Telomeres to ensure survival.
• A centromere to support segregation.
• An origin to initiate replication (see 12 The replicon).

All of these elements have been put together to construct a yeast artificial chromosome (YAC). This is a useful method for perpetuating foreign sequences. It turns out that the synthetic chromosome is stable only if it is longer than 20 V50 kb. We do not know the basis for this effect, but the ability to construct a synthetic chromosome offers the potential to investigate the nature of the segregation device in a controlled environment (Murray and Szostak, 1983).

This section updated 1-9-2000