Polyphenylalanine

LI 2 treatment Ribosome binding (pmol/pmol ribosome) Polyphenylalanine synthesis (pmol) Acetylation (pmol)... 

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. 

In the absence of an AGAC the ribosomes will prodnce the artificial polypeptides, polyphenylalanine (as specified by the codon UUU) or polyproline (as specified by the codon CCC). However, when streptomycin is added, the ribosomes prodnce a mixture of polythreonine (codon ACU) and poly serine (codon UCU). The misreading of the codons does not appear to be random U is read as A or C and C is read as A or U. If such misreading occurs in whole cells the accumulation of non-functional or toxic proteins would eventually prove fatal to the cells. There is some evidence that the bacterial cell membrane is damaged when the cells attempt to excrete the faulty proteins. 

If poly(U)-dependent polyphenylalanine synthesis is to be measured, the amount of template to be used is 5 fig/reaction mixture, the Mg acetate concentration is increased to 12 mM, and the amino acid mixture is omitted. 

Polyuridylic acid is a polynucleotide which can be used as a synthetic template RNA in a polypeptide synthesizing system (see Glossary for a discussion of these systems). It directs the incorporation of phenylalanine, and the product with poly U as template is polyphenylalanine. It has been observed that prior irradiation of the poly U template reduces its activity as a template.79,80 The amount of phenylalanine incorporation decreases with increasing dose of ultraviolet light, and some serine begins to become incorporated in the polypeptide chain. 

USA 47 1588-1602, 1961. The landmark paper reporting the finding that poly(U) stimulates the synthesis of polyphenylalanine. 

Nirenberg and Matthaei reported that polyuridylic acid codes for polyphenylalanine. 

Several other tj s are available, which employ electrostatic or hydrophobic interactions or metal chelate formation including polyarginine (79), polyphenylalanine or polycysteine (80), and dipeptide His-Trp (81). 

Another problem that may arise is that the protein fusion may lead to inactivation of the enzyme. The destabilization of P-galactosidase when using a polyphenylalanine lag is an illustradve example (80). There may also be interference between SH groups on the protein and cysteine groups on the fused affinity tail. 

Results to 3 One method giving polyphenylalanines (19) starts from the diazide (15 o) and others reacting with 4-benzyl-24 dioxo-oxozolidinc in pyridine (Eq. 9). Polypeptides containing the porphyrins in various ratxK are obtained. Now from stereoisomeric N-carbonic acid anhydrides of phenylalanines the D-, L- and DJL-fonns of fte polyphenylalanines were prepared. 

Fig. 4. Kinetics of polyphenylalanine synthesis at different temperatures, and Arrhenius plots of poly(phe) synthesis in S. solfataricus and E. coli poly-U programmed cell-free systems. Rates of polyphenylalanine synthesis were calculated from the initial slopes of the incorporation kinetics at different temperatures [Cammarano, P, unpublished results].

Optimal conditions for polyphenylalanine synthesis and tightness of subunit interaction in archaea ... 

Effect of aminoglycoside antibiotics on the fidelity and efficiency of polyphenylalanine synthesis in... 

Altamura et al. [178] and Londei et al. [179] investigated the ability of SOS and 30S subunits from phylogenetically disparate archaea to form synthetically active hybrid ribosomes with subunits from bacteria and eucarya, in the presence of Mg " concentrations (lS-18mM) which are optimal for polyphenylalanine synthesis. With poly(U) as the template and Phe-tRNA (or [jV-acetyl-Phe]-puromycin) as the substrate, SOS and 30S subunits from Euryarchaeota (M vannielii) and Crenarchaeota (5. solfataricus) could be assembled into hybrid active monosomes in all reciprocal combinations surprisingly, however, both reciprocal combinations of archaeal (S. solfataricus, M. vannielii) and eucaryal S. cerevisiae) ribosomal subunits gave rise... 

Ribosomes of Plasmodium knowlesi were isolated and characterized recently by Sherman et al. 21 These ribosomes sedimented in the 80S range and could be dissociated into 60S and 40S subparticles. The ribosomal RNA had a low % G+C of 37% and had sizes of 24.2S and 16.6S.22 The ribosomes demonstrated high activity in poly(IJ)-directed synthesis of polyphenylalanine and were strongly inhibited by 10 4m of nucleocidin, chlortetracycline, ethidium, puromycin, cycloheximide or berenil.23 Similar studies have been also carried out on Plasmodium lophurae, and similar profile of drug sensitivities were demonstrated.24 Most of the well-known antimalarial drugs tested showed no significant inhibitory activity in this in vitro assay. 

EF)T, (30) supports polyphenylalanine synthesis poorly. It is hydrolysed to guanosine-3, 5 -dipyrophosphate (31) by EF G and ribosomes. A chemical synthesis of (31) has been described. ... 

Sparsomycin, a sulfur-containing antibiotic, inhibits protein synthesis in mammalian and bacterial cells. Tryptophan administration before or after sparsomycin did not affect the hepatic polyribosomal disaggregation or the decreased protein synthesis due to sparsomycin.188 A possible explanation for the lack of effect by tryptophan may be due to sparsomycin s ability to cause fall-off ribosomes, which are defective as indicated by the decreased formation of polyphenylalanine when assayed in vitro with poly(U).207... 

Evidence also indicates that 16-membered-ring macrolides, as peptidyltransferase inhibitors, hinder the polyuridylic acid-dependent polymerization of phenylalanine, despite the fact that 14-membered-ring macrolides are not able to inhibit polyphenylalanine synthesis. In particular, 16-membered-ring macrolides containing at least one disaccharide-monoglycoside in their structures, such as leucomycin, spiramycin, carbomycin, and tylosin, may cause degradation of polyribosome [93,94]. 

Scientists set out to determine the specific codons for each amino acid. In 1961, Marshall Nirenberg prodnced the first crack in the genetic code (the collection of codons that specify all the amino acids fonnd in proteins). He showed that poly(U), a polynucleotide in which all the bases are uracil, produced polyphenylalanine in a cell-free protein-synthesizing system. Thus, UUU must be... 

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