Methods for studying mitochondrial transcription termination with isolated components.
Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.
Polosa
Paola Loguercio
PL
Dipartimento di Biochimica e Biologia Molecolare Ernesto Quagliariello, Università degli Studi di Bari, Bari, Italy.
Deceglie
Stefania
S
Roberti
Marina
M
Gadaleta
Maria Nicola
MN
Cantatore
Palmiro
P
D000818:Animals; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008928:Mitochondria / Q000201:enzymology*; D012617:Sea Urchins; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; C119291:mitochondrial D-loop-binding protein, Paracentrotus lividus; D012321:DNA-Directed RNA Polymerases