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Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein
Authors:R A Kastelein  B Berkhout  G P Overbeek  J van Duin
Institution:Department of Biochemistry, University of Leiden, Wassenaarseweg 64, 2333 AL LeidenThe Netherlands Tel. (0)71-148 333 ext. 5346
Abstract:The translational efficiency of the coat protein gene of phage MS2 has been examined in vivo with respect to neighbouring sequences. The cloned MS2 DNA has been gradually shortened starting at the 5' or 3' terminus, and its effect on coat protein synthesis monitored. Removal of the 3'-terminal sequences had little influence. In contrast, the gradual removal of the 5'-terminal region profoundly reduces translation. Long before the ribosomal binding site (RBS) of the coat protein (CP) gene is reached, the yield of CP has dropped by one order of magnitude. Functional half-lives of the various messengers were found not to be significantly different. Available evidence indicates that the secondary structure of the RBS in native and shortened MS2 RNA is identical. We infer that important determinants for ribosome recognition lie 5' to the RBS region of the MS2 RNA coat gene.
Keywords:RNA bacteriophage  recombinant DNA  mRNA structure  Shine-Dalgarno sequence  messenger half-life  AMV  alfalfa mosaic virus  BSA  bovine serum albumin  CP  coat protein  DMSO  dimethylsulfoxide  RBS  ribosomal binding site  SD sequence  Shine and Dalgarno sequence  SDS  sodium dodecylsulphate  TCA  trichloroacetic acid
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