Volume 1

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    DEAE paper, developed by Sanger et al. (1965) and Sanger and Brownlee (1967). Improvements of the system by Brownlee et al. (1968) led to its ability to separate oligonucleotides of up to twenty five residues in length. Molecules of RNA may therefore be digested with T1 ribonuclease to yield large fragments, which after their isolation are in turn digested with pancreatic ribonuclease into small fragments whose content is determined. Partial digestion with snake venom phosphodiesterase, which degrades polynucleo tides stepwise from the 3' terminus, can be used to establish most of the sequence, but does not reveal the order of the two or three nucleotides at the 5' terminus. Micrococcal nuclease may be used to release a dinucleotide from the 5' terminus. Transfer RNA is the smallest cellular RNA, sedimenting at about 4S in both bacteria and eucaryotic cells. The molecules at present sequenced vary in length from seventy-five to eighty-five nucleotides. The first tRNA to be sequenced was the alanine accepting species of yeast studied by Holley et al. (1965); transfer molecules for virtually all amino acids in a large number of species have since been determined and many of these sequences are shown in the figures of this chapter. Clover Leaf Model for Secondary Structure Although each tRNA has a distinct sequence and there are few bases in identical positions in all tRNA molecules, their overall secondary structures all fall into the same pattern. One feature of all the primary sequences is that no tRNA shows long complementary stretches which could allow base pairing —the greatest extent of duplex formation is usually seven base pairs. To achieve even this length, it is often necessary to include G-U base pairing as well as the more common G-C and A-U pairs. Secondary structures can be written for tRNA sequences by taking into account possible duplex formation between different parts of the molecule. Figure 5.1 shows the sequence of alanine tRNA written as a hairpin or a double hairpin; figure 5.2 shows the structures of several tRNAs, including tRNA ala of yeast, written as clover leafs. The double hairpin and clover leaf structures appear more stable because they rely upon longer segments of duplex RNA. These models can be distinguished by the different predictions which they make for the susceptibility of the bases at different positions to chemical or enzymic attack. Most ribonucleases attack bonds in single stranded regions preferentially and the points at which the chain should be split are different in the three models. And once bonds have been split, the different models would retain their structures to different extents; the hairpins would tend to separate into fragments rather easily, whereas its hydrogen bonded structure should make the clover leaf more resistant. Another approach is to study the avail ability of the different parts of the molecule to chemical mutagens; bases in the