Evidence for two RNA polymerase activities in Euglena gracilis chloroplasts.

B M Greenberg,J O Narita,C Deluca-Flaherty,W Gruissem,K A Rushlow,R B Hallick

doi:10.1016/s0021-9258(17)42686-x

Abstract

Two types of RNA polymerase activity were isolated from Euglena gracilis chloroplasts and compared. One polymerase is tightly bound to chloroplast DNA; this complex is called the transcriptionally active chromosome (TAC) (Rushlow, K. E., Orozco, E. M., Jr., Lipper, C., and Hallick, R. B. (1980) J. Biol. Chem. 255, 3786-3792). The other activity is found in a soluble extract of Euglena chloroplasts. The soluble extract is dependent upon an exogenous DNA template for activity. The two activities can be isolated in two distinct subchloroplast fractions from a single chloroplast preparation. The soluble extract is selective for transcription of transfer RNA genes, whereas the TAC is selective for ribosomal RNA genes. TAC and the soluble extract respond differently to KCl and Mg2+. The soluble extract is sensitive to heparin, and TAC is resistant. The two activities have different temperature optima. Based on this evidence, we conclude that Euglena chloroplasts have at least two distinct RNA polymerase activities.

Highlights

The other activity is found in a soluble extract both TAC and the soluble extract have alsobeenisolated of Euglena chloroplasts
The soluble extract is selective for transcription of transfer RNA genes, whereas the TAC is selective for ribosomal RNA genes
The production of tRNAs. 32P-labeled RNA from the tRNA With all sevenplasmid DNA templates containinEguglena region of the gel was elutedand hybridized torestriction chloroplast tRNA genes that are outside the rRNA operons endonucleasedigests of Euglena chloroplast DNA(Fig. 3, the soluble extract produces tRNA-sizedproducts (Fig. 4, RNA Polymerase Activities of Euglena gracilis Chloroplasts lunes 2-8)

Summary

Evidence forTwo RNA Polymerase Activities in Euglena gracilis Chloroplasts*

Based on this evidence,we conclude that Euglena chloand the soluble extract RNA polymerase activities directly. Our previous report[12]on isolation of a soluble transcription extract from Euglena was based on differential centrifhave all been located on chloroplast DNA restriction maps. Chloroplasts purified by flotation on sucrose [32] were resuspended in 3 volumes of 10 mM Tris-HC1 (pH 7.9), 1mM EDTA, 5mM dithiothreitol, frozen well understood.rRNA,tRNA,andmRNA gene loci have on dry ice, and thawed. Restriction maps for the Euglem chloroplast DNA templates are shown in Fig. In Vitro Transcription and RNA Analysis-RNA was transcribed from TAC as described by Rushlow et al [11, 33]. For transcription reactions involving the Euglena chloroplast soluble extract, a- reaction cocktailwas made containing 500 p~ ATP, 500 FM CTP, 50 I . The drugs were added to the reaction cocktail prior to addition of the soluble extract

RESULTS

Chloroolast DNA

Soluble Extract

ABC silly

DISCUSSION