Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics*

AUTOR(ES)

Levinger, Louis

FONTE

American Society for Biochemistry and Molecular Biology

RESUMO

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 ααββ 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 (α4–α5) 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 β10–β11 loop.

Documentos Relacionados

• The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity
• Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein
• Intracellular mRNA cleavage by 3′ tRNase under the direction of 2′-O-methyl RNA heptamers
• Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis
• Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL