Sequence of the 3′ end of the rRNA coding region from strain W3031a. Arrows indicate the approximate location of the 3′ end of mature 25S rRNA (−1), the site of PolI termination at the Reb1p terminator (+93), the approximate site of termination at the fail-safe terminator (+250), and the positions where probes 1 and 2 diverge from the rDNA sequence. Probe 1, used for S1 nuclease analysis of RNA 3′ end formation, is a single-stranded DNA complementary to the above sequence from position −72 (where the end label is incorporated) to position +179. Probe 2 is similar to probe 1 but complementary to the above sequence from −72 to +415.

Quantitation of transcript abundance by RT-PCR. (A) Locations of primers used for RT-PCR analysis of PolI termination. Also illustrated is the location of a 75-bp insert used to make an internal standard DNA for quantitative PCR. (B) Qualitative RT-PCR analysis of PolI transcript abundance. As described in Materials and Methods, primers 1 through 4 were individually used to make cDNA from equal aliquots of total yeast RNA. A fifth mock cDNA reaction in which no primer was added was run. An equal aliquot of each cDNA was then amplified by using the relevant primer pair (i.e., P1-P5 was used to amplify P1 cDNA, etc.). An sample was removed from each PCR every five cycles to measure the progress of amplification. As a control to show that each primer pair was equally active, each primer pair was also used to amplify rDNA. (C) Quantitative RT-PCR analysis of PolI transcript abundance. Aliquots of cDNA made with primers P1 through P4 (plus a blank cDNA in which no primer was added) were mixed with known amounts of an internal standard DNA and amplified with the appropriate primer pair. Each aliquot of cDNA corresponded to the amount of cDNA made from 0.15 μg of total yeast RNA. After separation by gel electrophoresis, amplification products were quantitated on a PhosphorImager.

Analysis of PolI termination in vivo on a ribosomal minigene, 105LT. (A) Structure of 105LT. The minigene consists of a PolI promoter (with an entire rDNA intergenic spacer upstream) attached to a 285-bp minigene. The sequence of the terminator fragment (C) consists of a 53-bp fragment with 23 bp of T-rich sequence upstream of the termination site at +285 and 30 bp including the Reb1p binding site downstream of termination. Probe 3, a single-stranded, end-labeled DNA used for S1 protection studies, is 322 nt long and diverges from the minigene sequence 247 nt from the labeled terminus. The +285 termination site on 105LT corresponds to position +93 on the chromosomal rDNA sequence shown in Fig. 1. (B) S1 nuclease analysis of PolI termination on 105LT, using probe 3. Lane 1, analysis of RNA from untransformed W3031a. No protected bands are seen other than undigested probe (322 nt). Lanes 2 and 3, RNA from two independent events transforming 105LT into into W3031a. A protected band of 247 nt corresponds to 3′ ends mapping to position +285 on 105LT, which in turn corresponds to position +93 on chromosomal rDNA. Bands below 247 nt presumably are due to 3′ processing of terminated transcripts. A band at 284 nt is at the location where probe sequence diverges from the minigene sequence and thus represents readthrough. Lane 4, same as lanes 2 and 3 except that the 105LT minigene contains a two-base substitution in the Reb1p site that abolishes Reb1p binding (mutant 1 panel C). As expected from in vitro studies, lack of Reb1p binding abolishes termini in the 247-nt region and lower and causes an increase in readthrough. Lane 5, same as lanes 2 and 3 except that the 105LT minigene contains a three-base substitution in the Reb1p binding site that increases the affinity for Reb1p about 10-fold (mutant 2 in panel C). As expected, increasing the affinity for Reb1p decreases the amount of readthrough relative to terminated RNA. Lane 6, same as lanes 2 and 3 except that the upstream element of the Reb1p terminator contains a three-base substitution which has been shown by in vitro experiments to impair release of paused transcripts (mutant 3 in panel C). This alteration of the release element causes an increase in the amount of primary termination product (possibly still paused and attached to the transcript) and a decrease in the amount of processed termini (probably released transcripts). Lane 7, S1 nuclease protection of an artificial readthrough transcript generated by T7 RNA polymerase transcription. This control verifies that the band at 284 nt in lanes 2 to 6 is due to readthrough transcription. Lane 8, S1 protection of a T7 transcript of a 105LT template that has been cut with BfrI. This transcript is expected to be 3 nt longer than the primary termination product at the Reb1p terminator. (C) Sequences of terminator mutants analyzed in panel B.

(A) S1 nuclease analysis of RNA 3′ ends formed on the chromosomal rDNA of strain W3031a. In lane 1, probe 1 was hybridized to total cellular RNA and digested with S1 nuclease, and the protected fragments were displayed on a denaturing acrylamide gel. The most prominent band is a cluster corresponding to the mature 3′ end of 25S rRNA (−1). The next most prominent bands are in a cluster extending from +93 downward to about +72. These bands are consistent with PolI termination at +93 followed by 3′ end processing of the released transcripts. A minor band at +179 gives an estimate of the amount of transcription that reads past the Reb1p terminator. Lane 2 is the same as lane 1 except that E. coli tRNA was added instead of yeast RNA. Lane M, DNA size markers (pBR322, HpaII digest; sizes indicated in nucleotides). (B) High-resolution mapping of RNA 3′ ends at the Reb1p terminator. The plasmid used to make probe 1 was digested with EcoRI and transcribed with T7 RNA polymerase to yield a runoff transcript with a 3′ terminus at +102. Lanes 1 and 2, probe 1 and the T7 transcript hybridized and digested with S1 nuclease as described in Materials and Methods except that the temperature was raised to 37°C to generate a ladder of bands extending downward from the full-length protected fragment. Lane 3, total yeast RNA hybridized with probe 1 and S1 nuclease digested at 30°C as described in Materials and Methods. Lane M is as in panel A. Note that the longest band due to PolI-terminated RNA is 9 nt shorter than the longest band due to the T7 transcript.

S1 nuclease analysis of RNA 3′ ends formed on the chromosomal rDNA of strain HI227 (rnt1-47). (A) Total cellular RNA was extracted from HI227 cells grown either at 24°C (lane 1) or at intervals after a shift to 37°C (lanes 2 to 4). Each RNA was hybridized with probe 1 and digested with S1 nuclease, and the protected fragments were displayed on a denaturing acrylamide gel. term, terminator. (B) Quantitation of the gel shown in panel A. Bands in the 3′ 25S rRNA region, in the +93 to +72 Reb1p terminator region, and at +179 were quantitated with a PhosphorImager, and their relative intensity is plotted as a function of time after the cells were shifted to 37°C. (C) Location of a fail-safe terminator. RNA from rnt1-47 cells grown for 4.5 h at 37°C was hybridized with probe 2 and analyzed by S1 nuclease protection. In addition to bands at −1 and +93 to +72, an additional cluster of bands is seen with a leading edge at about +250. Shorter bands are presumed to be 3′ processing products. Readthrough beyond the +250 site is negligible. Sizes are indicated in nucleotides.