Nucleotide Methylation in Ribosome Maturation

Summary

Principal Investigator: JASON RIFE
Abstract: Ribosome structure is remarkably conserved throughout all forms of life. However, it appears that biogenesis of these macromolecular machines is remarkably distinct in E. coli and S. cerevisiae, the two most common model systems. Despite the overall differences that exist in the two biogenic pathways there appears to at least a single point of commonality, which may provide a means to link these processes. All organisms likely contain a methyltransferase that almost always modifies two adjacent adenosines into N6,N6-dimethyladenosine within the terminal loop of all small ribosomal subunit RNA; in rare cases only a single adenosine is converted. This unique conservation of an RNA modification enzyme extends to the level of mitochondria and chloroplast ribosome biogenesis, as well. In E. coli this enzyme is termed KsgA, and Dim1 in S. cerevisiae. Investigation of this family of enzymes will reveal insights into the fundamental conservation of ribosome biogenesis. The long-term objective of this project is to understand the contribution this family of enzymes makes in the process of ribosome maturation at the molecular level. The specific aims of this project are to identify the minimal substrate for KsgA, detail the binding interaction of KsgA to 30S ribosomal subunits, and to probe the structure, function, mechanism, and regulation of the dimethylase activity of KsgA. A host of biochemical assays with KsgA, such as activity assays on reconstituted, incomplete 30S and chemical probing of the complex, will address issues of substrate recognition. Mutagenesis and x-ray crystallographic analysis of KsgA and Dim1 will be used to probe the structure and function of both proteins. Ultimately, these studies will reveal molecular details of methyltransferase reactions as well as likely provide the first evidence for a universal step in ribosome biogenesis.
Funding Period: 2005-02-01 - 2010-01-31
more information: NIH RePORT

Top Publications

  1. ncbi The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit
    Pooja M Desai
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298 0133, USA
    Arch Biochem Biophys 449:57-63. 2006
  2. ncbi Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly
    Pooja M Desai
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    Biochemistry 50:854-63. 2011
  3. ncbi Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design
    Heather C O'Farrell
    Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298 0133, USA
    Biochemistry 49:2697-704. 2010
  4. pmc Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases
    Nagesh Pulicherla
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, 23219, USA
    J Mol Biol 391:884-93. 2009
  5. pmc Scintillation proximity assay for measurement of RNA methylation
    Matthew R Baker
    Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298 0133, USA
    Nucleic Acids Res 37:e32. 2009
  6. pmc Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA
    Keith Connolly
    Department of Biology, University of Rochester, Rochester, NY 14627, USA
    Mol Microbiol 70:1062-75. 2008
  7. ncbi A conserved rRNA methyltransferase regulates ribosome biogenesis
    Zhili Xu
    Department of Biology, Hutchison Hall 301, University of Rochester, Rochester, New York 14627, USA
    Nat Struct Mol Biol 15:534-6. 2008
  8. ncbi Breaking the cycle of translation
    Jason P Rife
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
    Mol Cell 28:517-9. 2007
  9. pmc Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution
    Heather C O'Farrell
    Department of Biochemistry, Virginia Commonwealth University, Richmond 23298 0133, USA
    RNA 12:725-33. 2006
  10. pmc The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway
    Tamara Zarubica
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    RNA 17:346-55. 2011

Detail Information

Publications10

  1. ncbi The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit
    Pooja M Desai
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298 0133, USA
    Arch Biochem Biophys 449:57-63. 2006
    ..Therefore, it appears that KsgA solely senses the conformation 16S rRNA when carrying out its enzymatic activity...
  2. ncbi Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly
    Pooja M Desai
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    Biochemistry 50:854-63. 2011
    ..Therefore, the same technique of generating mutant 30S subunits can be used to study ribosome biogenesis on the whole...
  3. ncbi Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design
    Heather C O'Farrell
    Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298 0133, USA
    Biochemistry 49:2697-704. 2010
    ..We also discuss the inability thus far to determine a structure of a target adenosine bound in its active site...
  4. pmc Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases
    Nagesh Pulicherla
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, 23219, USA
    J Mol Biol 391:884-93. 2009
    ..Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures...
  5. pmc Scintillation proximity assay for measurement of RNA methylation
    Matthew R Baker
    Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298 0133, USA
    Nucleic Acids Res 37:e32. 2009
    ..This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format...
  6. pmc Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA
    Keith Connolly
    Department of Biology, University of Rochester, Rochester, NY 14627, USA
    Mol Microbiol 70:1062-75. 2008
    ..These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family...
  7. ncbi A conserved rRNA methyltransferase regulates ribosome biogenesis
    Zhili Xu
    Department of Biology, Hutchison Hall 301, University of Rochester, Rochester, New York 14627, USA
    Nat Struct Mol Biol 15:534-6. 2008
    ..These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation...
  8. ncbi Breaking the cycle of translation
    Jason P Rife
    Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
    Mol Cell 28:517-9. 2007
    ..Now, in this issue, the Green research group establishes unexpected complexity in decoding translation stop codons (Youngman et al., 2007)...
  9. pmc Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution
    Heather C O'Farrell
    Department of Biochemistry, Virginia Commonwealth University, Richmond 23298 0133, USA
    RNA 12:725-33. 2006
    ..This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life...
  10. pmc The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway
    Tamara Zarubica
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    RNA 17:346-55. 2011
    ..From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit...