BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 27. PROTEIN SYNTHESIS

27.8. Ribosomes, the Organelles of Protein Synthesis, Consist of Large and Small Subunits

Let us now turn to The Mechanism of Protein Synthesis. This complex process takes place in Ribosomes, which can be regarded as the Organelles of protein synthesis, much as Cell/35.html">Mitochondria are considered the organelles of Oxidative Phosphorylation. The ribosome is a highly specialized and intricate Structure with a diameter of approximately 200 Å. Ribosomes from E. coli have been studied in the greatest detail. Such a ribosome has a mass of 2500 kDa and a sedimentation coefficient of 70S. It can be dissociated into a large (50S) and a small (30S) subunit. These subunits can, in turn, be dissociated into their constituent Proteins and RNA molecules. The 30S subunit contains 21 proteins and a single molecule of 16S-RNA. The 50S subunit contains approximately 34 proteins and 2 RNA molecules (23S and 5S). An E. coli ribosome consists of roughly two-thirds RNA and one-third protein.

The cytoplasmic ribosomes of Eukaryotic Cells are somewhat larger than bacterial ribosomes. An intact eukaryotic ribosome has a sedimentation coefficient of 80S. Like its bacterial counterpart, it dissociates into a large (60S) and a small (40S) subunit. The small subunit contains one molecule of 18S-RNA, whereas the large subunit contains three RNA molecules (28S, 7S¹⁾, and 5S). Mitochondria and chloroplast ribosomes differ from Eukaryotic cytoplasmic ribosomes; they closely resemble 70S rather than 80S particles. Furthermore, Protein synthesis in mitochondria, METABOLISM/14.html">Chloroplasts, and Bacteria shares many features in common.

¹ By convention, this rRNA is more frequently designated as 5.8S-RNA. Transl. note.

Class="center">Fig. 27.11. Ribosomes can be dissociated into approximately 55 proteins and three RNA molecules

27.9. Ribosomes Can Be Reconstituted from Their Constituent Protein and RNA Molecules

The ribosomal 30S subunit can be reconstituted from a mixture of 16S-RNA and its 21 constituent proteins. The assembly of these components into a functionally active 30S subunit was first accomplished by Masayasu Nomura in 1968. A few years later, the 50S subunit was also successfully reconstituted. These experiments were of profound significance in two respects. First, they demonstrated that all the information required for the assembly of this organelle is encoded within The structure of its components; no extraneous extraribosomal factors are needed. Thus, in vitro ribosome formation is a self-assembly process. Second, reconstitution can be used to determine whether a particular component is required for ribosome assembly or for its actual functioning. For instance, this approach helped identify the ribosomal component responsible for sensitivity to the antibiotic streptomycin (Section 27.20). Studies on 30S subunit reconstitution led to the following Conclusions.

Fig. 27.12. Separation of proteins from the ribosomal 50S subunit by two-dimensional Polyacrylamide gel Electrophoresis

Fig. 27.13. Polyacrylamide gel electrophoretic separation of proteins from the native ribosomal 30S subunit (left) and the reconstituted 30S subunit (right). The reconstituted subunit contains the same set of PROTEINS AS THE native one

1. 16S-RNA is essential for both assembly and function. This requirement is highly specific, as 16S-RNA from Yeast cannot replace 16S-RNA from E. coli. The precise role of ribosomal RNA molecules remains an area of intense research interest.

2. In the reconstitution assays, individual proteins were systematically omitted to test whether an intact 30S subunit could still form from the remaining 20 proteins and 16S-RNA. It was found that at least six specific proteins are strictly required for the assembly of the 30S particle.

3. The assembly of a functionally active 30S particle requires the majority of its constituent proteins. Consequently, the 30S subunit operates as a functionally integrated, cooperative structure.

27.10 Proteins Are Synthesized from the Amino Terminus to the Carboxyl Terminus

One of the earliest questions regarding the mechanism of protein synthesis was whether chains grow from the amino terminus to the carboxyl terminus or vice versa. A definitive answer was provided by Howard Dintzis using pulse-labeling experiments. Reticulocytes actively synthesizing Hemoglobin were incubated with ³H-leucine for brief periods, too short for complete chains to be finished. The hemoglobin synthesized under these conditions was separated into α- and β-chains and digested with Trypsin. The specific radioactivity of the resulting Peptides—defined as The ratio of their radioactivity to their total leucine content—was then determined. Peptides located near the carboxyl terminus in the native protein exhibited higher radioactivity than those near the amino terminus. A progressive increase in radioactivity was observed from the amino end to the carboxyl end (Fig. 27.14). The carboxyl terminus was the most heavily labeled because it was synthesized last. Therefore, the direction of chain growth is from the amino terminus to the carboxyl terminus.

Fig. 27.14. Distribution of ³H-leucine in the hemoglobin α-chain synthesized after a brief pulse-labeling incubation of reticulocytes. The higher radioactivity at the C-terminus compared with the N-terminus indicates that the C-terminus is synthesized last