Synthesis of internal re-initiation fragments of beta-galactosidase in vitro and in vivo

Manley, J. L. (November 1978) Synthesis of internal re-initiation fragments of beta-galactosidase in vitro and in vivo. J Mol Biol, 125 (4). pp. 449-66. ISSN 0022-2836 (Print)0022-2836 (Linking)

DOI: 10.1016/0022-2836(78)90310-8


Internal re-initiation polypeptides which are products of the lacZ gene of Escherichia coli have been synthesized in a DNA-directed cell-free protein synthesis system. Some of the properties of these protein fragments have been characterized. The de novo synthesized re-initiation proteins, unlike both in vitro synthesized wild-type β-galactosidase and nonsense termination fragments, are insoluble when synthesized at 37 °C, but soluble if synthesis takes place at 25 °C. The same re-initiation proteins which are made in vivo have been detected in vitro. Unlike their in vivo counterparts, which are degraded rapidly, the in vitro synthesized restart proteins are completely stable for at least one hour following their synthesis. Both in vivo and in vitro, the re-initiation proteins are not synthesized from DNA containing a wild-type Z gene, but require a specific nonsense mutation in order to be expressed. Furthermore, the location of the mutation within the Z gene is very important in determining whether or not re-initiation will occur at a given site. Several nonsense mutations which do not result in the synthesis of detectable amounts of restart protein in vivo produce specific re-initiation polypeptides in vitro. These restart proteins display many of the same properties as do those which are made both in vivo and in vitro: they are not made from wild-type DNA, and they are only made from DNA containing a specific nonsense mutation. One of these mutations is 118, which is an extreme polar mutation in vivo. Another is 545, which synthesizes a restart protein in vivo if, and only if, it is coupled with a secondary mutation, π(1). π(1) thus appears to be necessary for the synthesis of a particular re-initiation protein in vivo but unnecessary for the synthesis of the same protein in vitro. The efficiencies of re-initiation vary at the different sites, but in all cases are less than the initiation frequency at the start of the gene. The experiments thus show that when complicating factors, such as polarity and protein degradation, are eliminated, translational re-initiation can be detected at many sites in the lacZ gene.

Item Type: Paper
Uncontrolled Keywords: DNA, Bacterial Escherichia coli/enzymology/genetics Galactosidases/*biosynthesis Genetic Complementation Test Mutation Peptide Chain Initiation, Translational Peptide Fragments/*biosynthesis Temperature beta-Galactosidase/*biosynthesis
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
organism description > bacteria
CSHL Authors:
Depositing User: Matt Covey
Date: 15 November 1978
Date Deposited: 14 Dec 2016 21:47
Last Modified: 14 Dec 2016 21:47
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