Ribosomes poly -directed protein synthesis

Figure 6. Poly(U) directed protein synthesis with ribosomes from 6—10 and 23—24 month-old rat livers, as a function of concentration Phe, phenylalanine or correct" incorporation Leu, leucine or "incorrect incorporation Leu/Phe, incorrect / correct incorporation. Curves for young and old are virtually identical.

The protein was oxidized and reduced as described in Table 3. This table and the assays for binding of L12 to depleted ribosomes, poly(U>directed polyphenylalanine synthesis, and enzymatic acetylation of L12 to form L7 are from Caldwell and coworkers37. 

Biochemical and genetic experiments in yeast have revealed that the b poly(A) tail and its binding protein, Pablp, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pablp, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the b end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells. 

Information on archaeal translation is essentially based on poly(U)- and poly(UG)-programmed cell-free systems and on peptidyltransferase assay systems. Poly(U)-directed systems have been used to monitor the reconstruction of archaeal ribosomal subunits and the susceptibility of archaea to protein synthesis inhibitors. 

The question remains whether CAP under certain circumstances blocks protein synthesis at the 80 S ribosome. In a cell-free system of reticulocyte microsomes (Weisberger et al., 1964 Beard et oL, 1969) an inhibition of the association of mRNA and the 40 S ribosomal subunit was reported. In the reticulocyte system used 70-100% inhibition of [ Cjleucine incorporation was reported at 0.1 pmole/ml of CAP with either endogenous template RNA or small amounts of poly(U). These results could not be confirmed by Zelkowitz et al. (1968). We also found no striking inhibition of poly (U)-directed amino acid incorporation by CAP in messenger-depleted cell-free systems from rat liver or rat embryos (Uehlin et al, 1974). From all this it seems clear that generally CAP has no striking effect on protein synthesis at the 80 S ribosome level. But inhibition of mammalian microsomal protein synthesis under certain conditions cannot be ruled out. 

The next question is which of the 64 triplets code for which amino acid In 1961, Marshall Nirenberg provided a simple experimental approach to the problem based on the observation that synthetic polynucleotides direct polypeptide synthesis in much the same manner as do natural mRNAs. Nirenberg incubated ribosomes, amino acids, tRNAs, and appropriate protein-synthesizing enzymes. With only these components, there was no polypeptide synthesis. However, when he added synthetic polyuridylic acid (poly U), a polypeptide of high molecular weight was synthesized. Even more important, the synthetic polypeptide contained only phenylalanine. With this discovery, the first element of the genetic code was deciphered the triplet UUU codes for phenylalanine. 

In 1962 Speyer, Basilio and I proved the validity of a hypothesis by Spotts and Stanier according to which the difference between streptomycin-sensitive, resistant and dependent E. coli. resides in the structure of their ribosomes. We established that streptomycin inhibits poly(U) directed polyphenylalanine synthesis in a fractionated cell-free protein synthesizing system only if the ribosomes in the system are taken from streptomycin-sensitive cells. These results indicated that in sensitive cells streptomycin inteiieres with ribosome action. As subsequent studies revealed, streptomycin is only one among many antibiotics whose site of action is the ribosome. 

Indeed, poly (U-G) and poly (U-Aza G) direct the incorporation of the same amino adds (the spedfidty is not altered) into polypeptides, but poly (U-Aza G) is less efficient when added to a subcellular protein-synthesis system derived from E. coli. With codons containing two Aza G, the interaction between the amino-acyl-transfer RNA and the triplet is so weakened that the translation may be interrupted, giving incompl polypeptide chains, which remain attached to the ribosomes. Ibis has been demonstrated in cell-free systems derived from aza-guanine-treated B. cereus. This also explains the differences observed by Chantreime between the d ree of inhibition of global protein synthesis and that of spedfic enzymes. 

Crude ribosomes or preincubated ribosomes have been subjected to methods of further purification. Chromatography of ribosomes on DEAE-cellulose has not yet been tested for enzyme synthesis (Traub and Zillig, 1966). By washing ribosomes with 1 M NH Cl in a suitable buffer, a protein fraction is dissociated from the ribosomes which contains most of the initiation factors. Enzyme synthesis becomes dependent on the addition of this fraction (Table 3) The initiation factors can also be separated on DEAE-cellulose and added back individually to the enzyme synthesis mixture (unpublished). The ribosomal subunits can be further disintegrated and reconstituted (Traub and Nomura, 1968 Nomura and Erdmann, 1970). In vitro reconstituted 30S ribosomes synthesize polyphenylalanine under the direction of poly U and they are also active in enzyme synthesis (Egberts et al., 1972). RNase III is removed by a similar ribosome washing procedure and the system becomes dependent on the addition of RNase III (Hercules et al., 1974 and unpublished). 

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