Effect of changes in the flexible arm on tRNase Z processing kinetics.
tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3' end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured alphaalphabetabeta hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (alpha4-alpha5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the beta10-beta11 loop.
Levinger
Louis
L
Department of Biology, York College of the City University of New York, Jamaica, New York 11451, USA. louie@york.cuny.edu
Hopkinson
Angela
A
Desetty
Rohini
R
Wilson
Christopher
C
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers; D004330:Drosophila / Q000201:enzymology; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D005998:Glycine; D006801:Humans; D007700:Kinetics; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment
D017931:DNA Primers; D029721:Drosophila Proteins; D008715:Methionine; D004722:Endoribonucleases; C502293:tRNase Z, Drosophila; D005998:Glycine