Quantitative analysis of ribosome-mRNA complexes at different translation stages.
Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.
Shirokikh
Nikolay E
NE
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Alkalaeva
Elena Z
EZ
Vassilenko
Konstantin S
KS
Afonina
Zhanna A
ZA
Alekhina
Olga M
OM
Kisselev
Lev L
LL
Spirin
Alexander S
AS
D017931:DNA Primers; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D048348:Reverse Transcription; D012270:Ribosomes / Q000378:metabolism*
D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger