Yeast protein synthesis

In nucleotide-depleted yeast, protein synthesis and amino acid incorporation is dependent on and is proportional to the amount of added purines and pyrimidines, which serve as precursors for RNA (379). Conversely, in amino acid-depleted S. aureus, RNA and protein synthesis both are dependent on and proportional to the amount of added amino acids (411). Furthermore, as we have already seen, no synthesis of protein takes place in pyrimidineless mutants in the absence of the missing RNA precursors and, conversely, no synthesis of RNA takes place in amino acid auxotrophs in the absence of the missing amino acid (412-416). The dependence of RNA synthesis on the presence of amino acids was also established for animal systems (liver) by Munro and co-workers (4l7, 418). 

Apphcations include ka olin clay dewatering, separation of fish oils from press Hquor, starch and gluten concentration, clarification of wet-process phosphoric acid, tar sands, and concentrations of yeast, bacteria, and fungi from growth media in protein synthesis (14). 

Fujiki, M. Vemer, K. (1993). Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. J. Biol. Chem. 268, 1914-1920. 

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. 

Feedback inhibition of amino acid transporters by amino acids synthesized by the cells might be responsible for the well known fact that blocking protein synthesis by cycloheximide in Saccharomyces cerevisiae inhibits the uptake of most amino acids [56]. Indeed, under these conditions, endogenous amino acids continue to accumulate. This situation, which precludes studying amino acid transport in yeast in the presence of inhibitors of protein synthesis, is very different from that observed in bacteria, where amino acid uptake is commonly measured in the presence of chloramphenicol in order to isolate the uptake process from further metabolism of accumulated substances. In yeast, when nitrogen starvation rather than cycloheximide is used to block protein synthesis, this leads to very high uptake activity. This fact supports the feedback inhibition interpretation of the observed cycloheximide effect. 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

Hatfield GW, Gurman GA (1992) Codon pair utilization bias in bacteria, yeast and mammals. In Hatfield DL, Lee BJ, Pirtle RM (eds) Transfer RNA in protein synthesis. CRC Press, Boca Raton, chap 7... 

Figure 12.5 The structures for four tRNA molecules of yeast, (a) Alanyl-tRNA (b) phenylalanyl-tRNA (c) seryl-tRNA (d) tyrosyl-tRNA. The single letter designations identify the sequence of bases along the single chain. Note that several of these are unusual bases, most of which are methylated (Me). Note also the ACC sequence at the 3 terminus of each tRNA. This is the site to which amino acids are attached in the process of protein synthesis, as indicated. These tRNA molecules have a substantial amount of secondary structure created by formation of Watson-Crick base pairs. Finally, note that the anticoding triplet in the bottom loop is shown.

Beside this dermatoxic activity pederin (147) has various biological activities (92). When administered in appropriate doses to partially hepatectomized rats, this compound stimulates development of hepatic tissues. The inhibitory effect at the cellular level has been found in chicken heart fibroblast cultures, and mice embryo, dog kidney, HeLa, and KB cell lines. In plants, root growth of Lupinus albus is inhibited and mitosis in Allium cepa blocked at the metaphasic stage. Also, pederin (147) inhibits protein synthesis and growth of yeast cells. In addition, the treatment of rat ascites sarcoma with purified extracts of P. fuscipes produces almost complete regression. 

Eleven subunit-subunit interactions have been identified between the 40S and 60S subunits of the yeast ribosome and once the two ribosomal subunits have assembled, they form a communicating ensemble (Gabashvili et al. 1999, 2003 Spahn et al. 2001). For example, the PTC of the large subunit has to be coordinated with the decoding center of the small subunit. After all, the distance between the two most important functional sites of the ribosome is approximately 75 A (Nakamura and Ito 2003 Ma and Nussinov 2004). Interaction between these sites that are far apart can be achieved either via transmission of conformational changes within and between the subunits or via ribosome-associated factors connecting the different sites. Both principles operate during protein synthesis (Rospert 2004). 

Dresios J, Derkatch IL, Liebman SW, Synetos D (2000) Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable. Biochemistry 39 7236-7244... 

A major goal in recombinant DNA technology is the production of useful foreign proteins by bacteria, yeast, or other cultured cells. Protein synthesis depends upon both transcription and translation of the cloned genes and may also involve secretion of proteins from the host cells. The first step, transcription, is controlled to a major extent by the structures of promoters and other control elements in the DNA (Chapter 28). Since eukaryotic promoters often function poorly in bacteria, it is customary to put the cloned gene under the control of a strong bacterial or viral X promoter. The latter include the X promoter PL (Fig. 28-8) and the lac (Fig. 28-2) and trp promoters of E. coli. These are all available in cloning vehicles. 

While defects in protein XPD often cause typical XP symptoms, some defects in the same protein lead to trichothiodystrophy (TTD, brittle hair disease). The hair is sulfur deficient, and scaly skin (ichthyosis, Box 8-F), mental retardation, and other symptoms are observed.0 Like their yeast counterparts (proteins RAD3 and RAD25), XPB and XPD are both DNA helicases.0 They also constitute distinct subunits of the human transcription factor TFIIHP, which is discussed in Chapter 28. It seems likely that XPD is involved in transcription-coupled repair (TCR) of DNA.° °i-s This is a subpathway of the nucleotide excision repair (NER) pathway, which allows for rapid repair of the transcribed strand of DNA. This is important in tissues such as skin, where the global NER process may be too slow to keep up with the need for rapid protein synthesis. Transcription-coupled repair also appears to depend upon proteins CSA and CSB, defects which may result in the rare cockayne syndrome.13 0 4 11 Patients are not only photosensitive but have severe mental and physical retardation including skeletal defects and a wizened appearance. 

Yeast protein L30, which is not homologous to any bacterial protein, controls its own synthesis by a feedback inhibition at the mRNA splicing step. L30 binds to its own pre-mRNA near the 5 splice site, blocking completion of the spliceosome assembly (Chapter 28).159... 

Fig. 5. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of 15N-labeled yeast ubiquitin synthesized by wheat germ cell-free protein synthesis system. Left lane molecular weight markers. Middle lane yeast ubiquitin translation solution. Right lane wheat germ alone (control). Yeast ubiquitin is indicated by...

Fig. 7. H-15N heteronuclear single-quantum coherence (HSQC) spectrum of yeast ubiquitin synthesized by wheat germ cell-free protein synthesis system, not purified (0.1 vaM, 128 [tl] 512 [t2] complex points, 512 scans), obtained at the H resonance frequency of 500 MHz. Spectral widths are 1600 and 6250 Hz in FI and F2, respectively.

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