Implication of Arginyl Residues in mRNA Binding to Ribosomes (original) (raw)
1980, European Journal of Biochemistry
Modification of Esclzevichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.
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Arginine Cofactors on the Polymerase Ribozyme
PLoS ONE, 2011
The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.
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.
Studies on the modification ofEscherichia coli ribosomal protein L7/L12 by succinic anhydride
Experientia, 1982
Experientia 38 (1982), Birkhfiuser Verlag, CH-4010 Basel/Switzerland 241 tion of fixative; perfusion of these small fish was not attempted. The brain was then quickly excised, placed in Rossmans' fixative precooled to -35 ~ and kept at that temperature for 24 h. Brains were then passed through 3 changes of ethanol at 4 ~ each of 1 h duration. Following this, specimens were cleared in xylene and embedded in wax. This method is designated 'experimental' in the table. Sections from all blocks were cut at 7 gin, floated on water (standard method) or alcohol (experimental method) and stained by methods listed in the table. Hydration was avoided wherever possible in 'experimental' methods. A brain was fixed for electron microscopy 17 while the fish was anaesthetized.
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
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