Polypeptide synthesis with ribonuclease-digested ribosomes (original) (raw)
Related papers
Polypeptide synthesis in Escherichia coli
Journal of Molecular Biology, 1963
The poly U-directedt synthesis of polyphenylalanine in the cell-free system from Escherichia coli is used as a model system in which to investigate the interaction of messenger RNA with ribosomes. It is shown that both the 508 and the 308 ribosome are necessary for polypeptide synthesis. The ribosomes accept the messenger RNA under conditions in which the dominant form is the 708 particles, but the particles involved will dimerize more readily than the rest of the 70 8 particles. All of the synthetic activity of the poly U-ribosome mixture appears as a rapidly sedimenting complex, 140 to 200 s. This active complex depends upon RNA for its integrity and contains an amount of poly U consistent with one molecule for several ribosomes. Furthermore, all of the synthetic capacity of the usual crude extract from E. coli is in the form of rapidly sedimenting complexes in the 100 to 200 s range.
European journal of biochemistry / FEBS, 1978
Modification of Escherichiu coli ribosomes with N-bromosuccinimide (SucNBr) (molar ratio of reagent to ribosome equal to 10) is accompanied by 100 -200 %increase in poly(U)-directed polyphenylalanine synthesis, while no change in peptidyl transferase activity ('fragment reaction') was observed. The magnesium dependence of the modified ribosomes in polyphenylalanine synthesis (optimum at 11 mM) is different from that of untreated ones (optimum at 9 mM). Hybridization of SucNBr-treated and untreated subunits showed that stimulation is produced by modification of the 50-S subunit.
Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids
Biochemistry, 2015
In an earlier study, β 3-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate βamino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β 3puromycin employed for ribosome selection influences the regio-and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA CUA activated with each of four methyl-β-alanine isomers (1−4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio-and stereochemistry as the O-methylated β-tyrosine moiety of β 3-puromycin. Also conducted were a selection of clones that are responsive to β 2-puromycin and a demonstration of reversal of the regio-and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes
The Journal of biological chemistry, 1991
Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin.
1972
Abbreviations. RNA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine. Enzymes. T, ribonuclease (EC 2.7.7.26); pancreatic ribonuclease (EC 2.7.7.16). Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-em path length cell. tions. After grinding with alumina [a], the cell paste was extracted with 10 mM MgC1, rather than 0.1 mM, to maintain the ribosomes as 70-5 particles. These 70-5 particles were washed by spinning through 0.5 M NH4Cl, 10 mM MgCl,, 10 mM Tris-HC1 pH 7.6 (cf. [5]), and were then dissociated into subparticles by resuspending in 50mM KC1, 0.3 mM MgCl,, 10mM Tris-HC1 p H 7.6. The subparticles were separated in a zonal rotor as before [I], and the 30-5 ribosomes precipitated with ethanol. The precipitate was dissolved in 0.3 mM magnesium acetate, 10 mM Tris-HC1 pH 7.6, and dialysed against this buffer, or against 1 mM magnesium acetate, 10 mM potassium phosphate buffer pH 7.2. Ribosomes were labelled as before [l] with 14Clabelled amino acids (CFB 104) and [3H]uridine (Radiochemical Centre, Amersham), except that isotope input was increased t o give specific activities of approximately 450 counts x min-l x pg-l for 14Clabelled protein, and 2500 counts x min-l x pg-l for [SH]RNA. Separation of RNA Protein Fragments Radioactive 30-5 ribosomes were hydrolysed with ribonuclease T, or pancreatic ribonuclease (Sigma) for 4.5 h a t room temperature, in the Vo1.29, No.3,1972
Biochemistry, 1992
The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed a t the a-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the t-amino group of natural lysyl-tRNA, and each was used to nonenzymatically initiate peptide synthesis. The fluorescent alanyl-tRNAs containing an AAA anticodon were used to initiate polyserine (with a synthetic tRNASer) or polyalanine synthesis from a poly(uridy1ic acid) template. The fluorescent lysyl-tRNA was used to initiate polylysine synthesis from poly(adeny1ic acid). Changes in the fluorescence of the amino-terminal coumarin were examined to characterize the environment of the probe as the nascent peptides were extended. Protection from proteolysis and the binding of anti-coumarin antibodies or Fab fragments suggest that the amino terminus of each polypeptide is protected from interaction with proteins (M, > 28 OOO) until the peptides are extended to an average length of 40-50 residues; however, the fluorescence from the amino terminus of shorter nascent polyalanine and polyserine peptides was readily quenched by methyl viologen (M , = 257), indicating ribosomes do not shield the nascent peptide from molecules of this size. The data appear to indicate that polyalanine, polyserine, and polylysine are extended from the peptidyl transferase into a protected region of the ribosome such as a groove or tunnel but that this region is readily accessible to small molecules.
Analytical Biochemistry, 1979
Thermal shock of intact Escherichia coli, which destroys selectively the 30 S ribosome subunit (A. Weiss and M. Tal, 1973, Biochemistry 12, 4534-4540), has been investigated as a simple procedure to prepare large amounts of pure 50 S ribosome subunits. The structural and functional properties of the 50 S particles prepared from a thermally shocked RNase 1-strain ofE. coli appear to be very similar to those of 50 S ribosomes isolated on sucrose gradients after dissociation of 70 S ribosomes. There was no significant difference in the protein composition of the two ribosome preparations. LiCl-Extracted ribosome cores had similar compositions and their functional activities could be reconstituted with either the homologous or heterologous split protein fractions. The 23 S rRNA of 50 S ribosomes isolated from the RNase 1-strain was essentially intact after the heat treatment. In contrast ribosomes and core particles from RNase + strains had extensively degraded RNA and functional activities could not be reconstituted.