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Molecular and Cellular Biology, February 1999, p. 1595-1604, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transcription Termination and 3'-End Processing of
the Spliced Leader RNA in Kinetoplastids
Nancy R.
Sturm,
Michael C.
Yu, and
David A.
Campbell*
Department of Microbiology and Immunology,
University of California Los Angeles School of Medicine, Los
Angeles, California 90095-1747
Received 16 July 1998/Returned for modification 14 October
1998/Accepted 6 November 1998
Addition of a 39-nucleotide (nt) spliced leader (SL) by
trans splicing is a basic requirement for all trypanosome
nuclear mRNAs. The SL RNA in Leishmania tarentolae is a
96-nt precursor transcript synthesized by a polymerase that resembles
polymerase II most closely. To analyze SL RNA genesis, we mutated SL
RNA intron structures and sequence elements: stem-loops II and III, the
Sm-binding site, and the downstream T tract. Using an exon-tagged SL
RNA gene, we examined the phenotypes produced by a second-site 10-bp
linker scan mutagenic series and directed mutagenesis. Here we report
that transcription is terminated by the T tract, which is common to the
3' end of all kinetoplastid SL RNA genes, and that more than six T's
are required for efficient termination in vivo. We describe mutants
whose SL RNAs end in the T tract or appear to lack efficient
termination but can generate wild-type 3' ends. Transcriptionally
active nuclear extracts show staggered products in the T tract,
directed by eight or more T's. The in vivo and in vitro data suggest
that SL RNA transcription termination is staggered in the T tract and
is followed by nucleolytic processing to generate the mature 3' end. We
show that the Sm-binding site and stem-loop III structures are
necessary for correct 3'-end formation. Thus, we have defined the
transcription termination element for the SL RNA gene. The termination
mechanism differs from that of vertebrate small nuclear RNA genes and
the SL RNA homologue in Ascaris.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095. Phone: (310) 825-4195. Fax: (310) 206-3865. E-mail: dc{at}ucla.edu.
Molecular and Cellular Biology, February 1999, p. 1595-1604, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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