Showing papers by "Suparna Sanyal published in 2008"

A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli.

Abstract: With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3'-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.

Abstract: BACKGROUND: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. CONCLUSION/SIGNIFICANCE: 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

Abstract: The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.

Abstract: The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF‐Tu, which forms a ternary complex with aminoacyl(aa)‐tRNA. To study the binding modes of different aa‐tRNAs, we compared cryo‐EM maps of the kirromycin‐stalled ribosome bound with ternary complexes containing Phe‐tRNAPhe, Trp‐tRNATrp, or Leu‐tRNALeuI. The three maps suggest a common binding manner of cognate aa‐tRNAs in their specific binding with both the ribosome and EF‐Tu. All three aa‐tRNAs have the same ‘loaded spring’ conformation with a kink and twist between the D‐stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF‐Tu‐binding CCA end of aa‐tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa‐tRNA selection.

Role of the ribosome in protein folding.

Abstract: In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding.