Polyribosomes

Ben-Porat et al. (1971) showed that the larger polysomes that are characteristic of cells infected with PRV contained more RNA that would hybridize to viral DNA and a lower proportion of lysine in nascent polypeptides than the smaller polysomes of infected or uninfected cells. Using the declining ratio of lysine to leucine as a measure of the shut-off of host protein synthesis, they found that the decline was prevented by actinomycin added at the time of infection and arrested by actinomycin added at 3 hr after infection. The effect 

The early breakdown of polysomes that followed infection with HSV-2 was not prevented by cycloheximide (Fenwick and Walker, 1978) indicating that it did not depend on newly made protein, nor on continuing translation with its associated termination of polypeptide chains and detachment of ribosomes from the messenger. 

Silverstein and Engelhardt (1979) measured the protein-synthesizing activity of polysomes and rates of chain elongation of polypeptides, and found that the translation rate did not alter during the time that the polysomes were declining. They also concluded that a substantial proportion of the larger polysomes that formed after the early breakdown were inactive in protein synthesis and suggested a second block which resulted in inhibition of translation but not breakdown of polysomes and affected specifically cellular and early viral protein synthesis. 

The earliest reports of attempts to analyze the proteins made in infected cells by gel electrophoresis (Spear and Roizman, 1968 Shi-mono et ai, 1969) provided further evidence of the declining synthesis of cellular proteins and a concomitant rise in the production of new and different proteins, some of which were recognizable as virus 

Structural proteins. These changing patterns of protein synthesis were seen in clearer detail using autoradiography of dried polyacrylamide gels (Honess and Roizman, 1973). Honess and Roizman (1975) also used cycloheximide to confine viral mRNA synthesis to the production of immediate-early (a) mRNA. They found that, after reversal of cycloheximide, host protein synthesis declined but that the decline was less rapid if actinomycin was added at the same time as removing the cycloheximide, suggesting that p-polypeptides were at least partly responsible for the inhibition of cellular protein synthesis. 

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Bewley, J.D. Larsen, K. (1980). Cessation of protein synthesis in water-stressed pea roots and maize mesocotyls without loss of polyribosomes. Effects of lethal and non-lethal water stress. Journal of Experimental Botany, 32, 1245-56. 

Drought also has a profound effect on protein synthesis. In many plant tissues, a reduced water potential causes a reduction of total protein synthesis and a rapid dissociation of polyribosomes. The latter has been shown not to be the consequence of increase in ribonuclease activity (Hsiao, 1973 Dhindsa Bewley, 1976). For a specific protein, Jacobsen, Hanson Chandler (1986) have shown in barley leaves that water stress enhances the synthesis of one of the a-amylase isozymes. Using a cDNA probe they found that water-stressed leaves contained much more a-amylase mRNA than unstressed plants. 

Lin, C.Y. Key, J.L. (1967). Dissociation and reassembly of polyribosomes in relation to protein synthesis in the soybean root. Journal of Molecular Biology, 26, 237-47. 

Many ribosomes can translate the same mRNA molecule simultaneously. Because of their relatively large size, the ribosome particles cannot attach to an mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same mRNA molecule form a polyribosome, or polysome. In an unrestricted system, the number of ribosomes attached to an mRNA (and thus the size of polyribosomes) correlates positively with the length of the mRNA molecule. The mass of the mRNA molecule is, of course, quite small compared with the mass of even a single ribosome. 

A major sorting decision is made early in protein biosynthesis, when specific proteins are synthesized either on free or on membrane-bound polyribosomes. This results in two sorting branches called the cytosolic branch and the rough endoplasmic reticulum (R R) branch (Figure 46-1). This sorting occurs because proteins synthesized on membrane-bound polyribosomes contain a signal peptide that mediates their attachment to the membrane of the ER. Further details on... 

Figure 46-1. Diagrammatic representation of the two branches of protein sorting occurring by synthesis on (1) cytosolic and (2) membrane-bound polyribosomes. The mitochondrial proteins listed are encoded by nuclear genes. Some of the signals used in further sorting of these proteins are listed in Table 46-4. (ER, endoplasmic reticulum GA, Golgi apparatus.)...

Matrix proteins must pass from cytosolic polyribosomes through the outer and inner mitochondrial membranes to reach their destination. Passage through the two membranes is called translocation. They have an amino terminal leader sequence (presequence),... 

Translocation is believed to occur posttranslation-ally, after the matrix proteins are released from the cytosolic polyribosomes. Interactions with a number of cytosolic proteins that act as chaperones (see below) and as targeting factors occur prior to translocation. 

THE SIGNAL HYPOTHESIS EXPLAINS HOW POLYRIBOSOMES BIND TO THE ENDOPLASMIC RETICULUM... 

As indicated above, the rough ER branch is the second of the two branches involved in the synthesis and sorting of proteins. In this branch, proteins are synthesized on membrane-bound polyribosomes and translocated into the lumen of the rough ER prior to further sorting (Figure 46-2). 

The signal hypothesis was proposed by Blobel and Sabatini pardy to explain the distinction between free and membrane-bound polyribosomes. They found that proteins synthesized on membrane-bound polyribosomes contained a peptide extension (signal peptide)... 

Most proteins that are synthesized on membrane-bound polyribosomes and are destined for the Golgi apparatus or plasma membrane reach these sites inside transport vesicles. The precise mechanisms by which proteins synthesized in the rough ER are inserted into these vesicles are not known. Those involved in transport from the ER to the Golgi apparatus and vice versa—and from the Golgi to the plasma membrane— are mainly clathrin-free, unlike the coated vesicles involved in endocytosis (see discussions of the LDL receptor in Chapters 25 and 26). For the sake of clarity, the non-clathrin-coated vesicles will be referred to in... 

Many proteins are targeted to their destinations by signal sequences. A major sorting decision is made when proteins are partitioned between cytosohc and membrane-bound polyribosomes by virtue of the absence or presence of a signal peptide. 

Many proteins synthesized on membrane-bound polyribosomes proceed to the Golgi apparatus and the plasma membrane in transport vesicles. 

B. Plasma Proteins Are Generally Synthesized ON Membrane-Bound Polyribosomes ... 

Waxman HS, Rabinowitz M. 1966. Control of reticulocyte polyribosome content and hemoglobin synthesis by heme. Biochim Biophys Acta 129 369-379. 

The field of translation initiation has focused on the initial round ofribosomal subunit recruitment to an mRNA. Presumably, these events are mirrored in the subsequent rounds of initiation necessary for polyribosome formation. Importantly, because mRNAs are typically present in large polyribosomes (averaging 9-13 ribosomes per mRNA), the initiation events that govern ribosome recruitment to preexisting polyribosomes constitute the majority of translation initiation cycles occurring in an mRNA s lifetime. Whether or not these initiation events mimic the first round of initiation is not yet known. Since eukaryotic cells divide ribosomes between two subcellular compartments, the cytosol and ER membrane, it is also important to know if the mechanism of translation initiation on ER-bound ribosomes is similar to that occurring on soluble ribosomes and, importantly, whether ER-bound ribosomes can direcdy (re) initiate translation on bound polyribosmes. 

The methods described in this section can be used to examine the in vivo dynamics of polyribosome assembly on the ER membrane. Here, we will... 

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