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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    heavy to hybrid ribosomes takes place, at incubation temperatures of either 0°C or 37 3 C, showing that subunits are exchanged. An apparent exchange of subunits, however, can be generated by an artefact which makes density centrifugation of ribosomes unreliable. Infante and Baierlein (1971) found by centrifuging ribosomes of sea urchin eggs at high speeds that the distribution depends not only on ionic conditions but also upon the gravitational field and the ribosome concentration. Hydrostatic pressure seems to shift an equilibrium between ribosomes and subunits towards dissociation. The result of this shift is that the apparent sedimentation velocity of the ribosomes is changed; such changes have sometimes been interpreted as evidence that there are changes in the conformation of ribosomes at parti cular stages of protein synthesis, but these effects are instead an artefact of centrifugation. The effect of hydrostatic pressure on the heavy ribosomes is to mimic the formation of hybrid ribosomes. Fixation with glutaraldehyde before the ribosomes are centrifuged prevents dissociation in the gradient; for Subramanian and Davis (1971, 1973) showed that when heavy and light ribosomes are allowed to run off polysomes at 0°C and are fixed with glutar aldehyde before centrifugation, only two separate peaks of heavy and light ribosomes are found. The absence of hybrid ribosomes implies that their presence in experiments in which the fixation step is omitted represents an artefact induced by hydrostatic pressure. But when ribosomes are allowed to run off at 37°C, a peak of hybrid ribosomes is found, even when the fixation step is included. However, this peak can be produced just as readily whether the two sets of ribosomes are allowed to run off together or are run off in separate incubations and are then mixed ten minutes later. This means that although an exchange of subunits takes place, it occurs well after chain termination, presumably because incubation conditions promote a ready dissociation and reassociation of free ribosomes independent of protein synthesis. The temperature dependence of this effect suggests that it may be catalysed by the dissociation factor, whose action is reversible. In vitro experiments are therefore unable to reveal the state in which ribo somes are released from messengers under physiological conditions. At 0°C, ribosomes are released as 70S particles; and the presence of hybrid ribo somes results only from an artefact of centrifugation. But low temperature conditions may not be typical of those prevailing in the cell. At 37°C, subunits are exchanged to generate hybrid ribosomes in a process which does not depend upon protein synthesis; this therefore provides no information about the state in which they are released from the messenger. ■ A similar rapid equilibrium between 80S mammalian ribosomes released upon termination of protein synthesis and subunits present in the incubation has been demonstrated by Falvey and Staehelin (1970). The 80S subunits