T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis.

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  • Additional Information
    • Source:
      Publisher: BioMed Central Country of Publication: England NLM ID: 101088663 Publication Model: Electronic Cited Medium: Internet ISSN: 1472-6750 (Electronic) Linking ISSN: 14726750 NLM ISO Abbreviation: BMC Biotechnol Subsets: MEDLINE
    • Publication Information:
      Original Publication: [London] : BioMed Central, 2001-
    • Subject Terms:
      High-Throughput Screening Assays/*methods ; RNA/*analysis ; RNA Ligase (ATP)/*genetics ; RNA Ligase (ATP)/*metabolism ; Viral Proteins/*genetics ; Viral Proteins/*metabolism; Adenosine Monophosphate/chemistry ; Adenosine Monophosphate/metabolism ; Analysis of Variance ; Catalytic Domain ; Electrophoresis, Polyacrylamide Gel ; Hydrogen-Ion Concentration ; Mutation ; Polyethylene Glycols/chemistry ; RNA/genetics ; RNA/metabolism ; RNA Ligase (ATP)/chemistry ; RNA Ligase (ATP)/isolation & purification ; Viral Proteins/chemistry ; Viral Proteins/isolation & purification
    • Abstract:
      Background: T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA.
      Results: With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.
      Conclusions: Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.
    • References:
      Structure. 2004 Feb;12(2):327-39. (PMID: 14962393)
      Methods. 2008 Jan;44(1):3-12. (PMID: 18158127)
      Curr Opin Struct Biol. 2008 Feb;18(1):96-105. (PMID: 18262407)
      RNA. 2010 Dec;16(12):2537-52. (PMID: 20921270)
      Nucleic Acids Res. 1984 Nov 12;12(21):8235-51. (PMID: 6504699)
      Science. 2001 Oct 26;294(5543):858-62. (PMID: 11679671)
      Cell. 2006 Oct 6;127(1):71-84. (PMID: 17018278)
      Science. 2008 May 23;320(5879):1077-81. (PMID: 18403677)
      J Biol Chem. 2003 May 16;278(20):17601-8. (PMID: 12611899)
      RNA. 2009 Dec;15(12):2375-84. (PMID: 19861428)
      Curr Opin Cell Biol. 2009 Jun;21(3):377-86. (PMID: 19307111)
      Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12709-14. (PMID: 12228725)
    • Accession Number:
      0 (Viral Proteins)
      3WJQ0SDW1A (Polyethylene Glycols)
      415SHH325A (Adenosine Monophosphate)
      63231-63-0 (RNA)
      EC 6.5.1.3 (RNA Ligase (ATP))
      EC 6.5.1.3 (bacteriophage T4 RNA ligase 2)
    • Publication Date:
      Date Created: 20110705 Date Completed: 20111028 Latest Revision: 20211020
    • Publication Date:
      20221213
    • Accession Number:
      PMC3149579
    • Accession Number:
      10.1186/1472-6750-11-72
    • Accession Number:
      21722378