Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers.
Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase. Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts. Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts. Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts. Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function.
Kholod
N
N
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia.
Vassilenko
K
K
Shlyapnikov
M
M
Ksenzenko
V
V
Kisselev
L
L
D017123:Bacteriophage T7 / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D010444:Peptide Elongation Factor Tu; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins
D012360:RNA, Transfer, Phe; D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010444:Peptide Elongation Factor Tu