The Binding of Met‐tRNAf to Isolated 40‐S Ribosomal Subunits and the Formation of Met‐tRNAf· 80‐S‐Ribosome Initiation Complexes
Authors: SMITH Kelvin E. , RICHARDS Anne C. , ARNSTEIN Henry R. V. 
Published: March 2005

Different forms of 40‐S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40‐S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37‐g · CM−3 form is not present in the cell but the other two forms, the 1.40‐g · CM−3 and 1.40‐g · CM−3 subunits, are present in cell extracts. 35S label is bound to 1.37‐g · cm−3 and 1.40‐g · cm−3 subunits when [35S]MettRNAf, GTP and poly(A,U,G) are included in the incubations. The 35S‐labelled 40‐S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met‐tRNAf· 40‐S‐subunit · poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37‐g · cm−3 particle is no longer seen and the total quantity of 35S label on the 40‐S subunits is 90 % lower after sucrose gradient purification.Between 30 % and 40 % of the 40‐S subunits bind [35S]Met‐tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met‐tRNAf over derived 40‐S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit‐bound 35S label ultimately recovered. With these incubations less than 10% of the 40‐S subunits have bound [35S]Met‐tRNAf. [35S]Met‐tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9‐S globin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations codons determines the amount of 35S label bound. Similar results are obtained for the ribosomal‐wash‐dependent binding of [35S]Met‐tRNAf to 80‐S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9‐S globin mRNA but not poly(A,U,G) stimulates protein synthesis in the wheat germ system. HCHOtreated globin mRNA, although stimulatory, is 45 % less effective than native mRNA.The addition of AUG, derived 60‐S subunits and extra ribosomal wash is required for the formation of [35S]Met‐tRNAf . 80‐S‐ribosome complexes from sucrose‐gradient‐purified [35S]Met‐tRNAf· 40‐S‐subunit complexes. The 80‐S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80‐S ribosome. 35S label is released as methionyl‐puromycin.If the [35S]Met‐tRNAf · 40‐S‐subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60‐S subunits are required to form [35S]Met‐tRNAf· 80‐Sribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.