We have examined the effects of nucleosome cores on the initiation and 
elongation of RNA transcripts by phage T7 RNA polymerase in vitro. A 
transcription template, pT207-18, was constructed containing tandemly repeated 
207 base-pair (bp) nucleosome positioning sequences from a sea urchin 
(Lytechinus variegatus) 5 S RNA gene inserted between the T7 and SP6 
transcription promoters of pGEM-3Z. Nucleosome cores were reconstituted onto 
supercoiled, closed circular pT207-18 DNA and double label transcription 
experiments were performed to determine the effects of nucleosome cores on the 
initiation and elongation of transcripts by T7 RNA polymerase. Both transcript 
initiation and elongation were inhibited, the extent of the inhibition being 
directly proportional to the number of nucleosome cores reconstituted onto the 
pT207-18 DNA templates. Time course transcription experiments indicated that 
nucleosome cores caused a reduction in the equilibrium length of transcripts and 
not mere retardation of elongation rates. Continuous regularly spaced linear 
arrays of nucleosomes were obtained by digesting reconstituted nucleosomel 
pT207-18 templates with DraI, for which a unique restriction site lies within 
the nucleosome positioning region of the 207 bp 5 S rDNA repeat sequence. After 
in vitro transcription with T7 RNA polymerase an RNA ladder with 207 nucleotide 
spacing was obtained, indicating that transcription can occur through continuous 
arrays of positioned nucleosome cores. It is demonstrated that nucleosome cores 
partially inhibit the elongation of transcripts by T7 RNA polymerase, while 
allowing passage of the transcribing polymerase through each nucleosome core at 
an upper limit efficiency of 85%. Hence, complete transcripts are produced with 
high efficiency from short nucleosomal templates, while the production of 
full-length transcripts from long nucleosomal arrays is relatively inefficient. 
The results indicate that nucleosome cores have significant inhibitory effects 
in vitro not only on transcription initiation but on transcription elongation as 
well, and that special mechanisms may exist to overcome these inhibitory effects 
in vivo.