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Papers by Narasimham Jammi

Research paper thumbnail of Interaction of the 3\u27 end of tRNA with the ribosome: Methods and modifications of the 3\u27 end

The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ... more The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ribosome. The first step was to attach a thiol group on to the 3' end of tRNA. This could then be alkylated by iodacetamido, 1-10 orthophenanthroline. Phenanthroline, in the presence of copper and a reducing agent, cleaves nearby nucleic acids via an as yet unknown mechanism. However, the modification process proved to be a very difficult task. Various modification procedures were tried. 5' thiophosphorylation of tRNA proved to be unsuccessful. Ligation of a thiolated nucleotide to the 3' end of tRNA-A was also a failure. Introduction of a thiophosphate at the 3' end of tRNA was yet another procedure that was not able to be accomplished. Finally, annealing of two fi*agments to form a compact tRNA structure was also not acheived. In short, this manuscript explored various avenues for modification of the 3* end of tRNA. This should serve as a guide for future work in this field.

Research paper thumbnail of Interaction of the 3' end of tRNA with the ribosome: Methods and modifications of the 3' end

The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ... more The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ribosome. The first step was to attach a thiol group on to the 3' end of tRNA. This could then be alkylated by iodacetamido, 1-10 orthophenanthroline. Phenanthroline, in the presence of copper and a reducing agent, cleaves nearby nucleic acids via an as yet unknown mechanism. However, the modification process proved to be a very difficult task. Various modification procedures were tried. 5' thiophosphorylation of tRNA proved to be unsuccessful. Ligation of a thiolated nucleotide to the 3' end of tRNA-A was also a failure. Introduction of a thiophosphate at the 3' end of tRNA was yet another procedure that was not able to be accomplished. Finally, annealing of two fi*agments to form a compact tRNA structure was also not acheived. In short, this manuscript explored various avenues for modification of the 3* end of tRNA. This should serve as a guide for future work in this field.

Research paper thumbnail of Small molecule inhibitors of the RNA-dependent protein kinase

Biochemical and Biophysical Research Communications, 2003

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase t... more The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the a subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Research paper thumbnail of Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Nucleic Acids Research, 2001

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosph... more The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho-and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.

Research paper thumbnail of Identification of ribosome-ligand interactions using cleavage reagents

Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 1995

To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthr... more To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.

Research paper thumbnail of Interaction of the 3\u27 end of tRNA with the ribosome: Methods and modifications of the 3\u27 end

The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ... more The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ribosome. The first step was to attach a thiol group on to the 3' end of tRNA. This could then be alkylated by iodacetamido, 1-10 orthophenanthroline. Phenanthroline, in the presence of copper and a reducing agent, cleaves nearby nucleic acids via an as yet unknown mechanism. However, the modification process proved to be a very difficult task. Various modification procedures were tried. 5' thiophosphorylation of tRNA proved to be unsuccessful. Ligation of a thiolated nucleotide to the 3' end of tRNA-A was also a failure. Introduction of a thiophosphate at the 3' end of tRNA was yet another procedure that was not able to be accomplished. Finally, annealing of two fi*agments to form a compact tRNA structure was also not acheived. In short, this manuscript explored various avenues for modification of the 3* end of tRNA. This should serve as a guide for future work in this field.

Research paper thumbnail of Interaction of the 3' end of tRNA with the ribosome: Methods and modifications of the 3' end

The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ... more The goal of this Masters thesis project was to map the neighborhood of the 3' end of tRNA on the ribosome. The first step was to attach a thiol group on to the 3' end of tRNA. This could then be alkylated by iodacetamido, 1-10 orthophenanthroline. Phenanthroline, in the presence of copper and a reducing agent, cleaves nearby nucleic acids via an as yet unknown mechanism. However, the modification process proved to be a very difficult task. Various modification procedures were tried. 5' thiophosphorylation of tRNA proved to be unsuccessful. Ligation of a thiolated nucleotide to the 3' end of tRNA-A was also a failure. Introduction of a thiophosphate at the 3' end of tRNA was yet another procedure that was not able to be accomplished. Finally, annealing of two fi*agments to form a compact tRNA structure was also not acheived. In short, this manuscript explored various avenues for modification of the 3* end of tRNA. This should serve as a guide for future work in this field.

Research paper thumbnail of Small molecule inhibitors of the RNA-dependent protein kinase

Biochemical and Biophysical Research Communications, 2003

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase t... more The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the a subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Research paper thumbnail of Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Nucleic Acids Research, 2001

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosph... more The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho-and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.

Research paper thumbnail of Identification of ribosome-ligand interactions using cleavage reagents

Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 1995

To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthr... more To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.