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Spinach chloroplast RNA polymerase from crude lysates was
able to recognize bacterial histidine and threonineterminators.
To determine whether chloroplast RNA polymerase would terminate
other baterial terminators invitro,several terminators isolated
from the attenuators of E.coli amino acid operons, such as leu,
trp, ilvGEDA, phe and ilvB, were transcribed by spinach
chloroplast RNA polymerase and T7 RNA polymerase. The
termination efficiency of each terminater was quantitated with
laser densitometry. Transcription results reveal that the
chloroplast and T7 RNA polymerases did terminate transcription
at different terminators with varying efficiencies. The thr
terminator wass till observed as the most efficient terminator.
Previous studies with thr terminator mutants indicatedthat the
transcription termination efficiency of thrterminator by
spinach chloroplast RNA polymerase was modulated by a not-yet-
transcribed 5 bases ( CGACT ) DNA sequences. These 5 bases are
located immediately from the termination release site. To
further characterize the effect of these 5bases on termination,
a series of systematic mutants for these 5 bases were created
by PCR with a mixed primer. These thr variants were then
transcribed in vitro by chloroplast or T7 RNA polymerase.
Transcription by chloroplast RNA polymerase indicated that the
second base of these 5 bases is important for determining the
terminationefficiency.However, the transcription termination by
T7 RNA polymerase did not reveal any difference among these
mutants.These results suggested that the termination process of
chloroplast RNA polymerase is complex and differs from T7 RNA
polymerase.
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