1. Transcriptional Termination Factors for RNA Polymerase II in Yeast
Agustı́n Aranda, Nick Proudfoot 
Molecular Cell 
Volume 7, Issue 5, Pages (May 2001)
DOI: /S (01)

2. Figure 1 Northern Blot Analysis of Mutants in 3′ End Formation
(A) Diagram of the intron-based reporter gene. The ura4+ coding sequence is represented by white boxes, the poly(A) signal inside the intron is represented by a gray box, and the ura4+ pause element is represented by a striped box. All the possible RNAs produced by the reporter gene are indicated underneath.
(B) Northern blot analysis of cis mutants. The multicopy plasmids obtained by gap repair from the mutant strains were sequenced, and those harboring mutations were introduced into the wild-type A1 strain. Total RNA was extracted, separated in a 1.5% formaldehyde gel, transferred to nylon, and hybridized with an ura4+-specific HincII-EcoRV probe. The percentage of truncated (T) relative to total (T + WT) transcripts is indicated for each mutant. The extent of the deletion inside the reporter gene is described under each mutant.
(C) Northern blot analysis of various trans mutants. Dominant mutants are indicated by “M”, and recessive ones are indicated by “m”. The strains were transformed with the wild-type reporter plasmid pUIpA+C-F to increase the signal, and the blot was carried out as in (B).
(D) Loading control of WT and m70. After hybridizing with the ura4-specific probe, the filter was stripped and hybridized with an 18S rRNA probe (Hansen et al., 1998). The ratio of the ura4-derived transcripts relative to rRNA is indicated below each line, considering 1 the value of the WT
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Run-On Analysis of Some trans Mutants
The diagram shows the reporter vector pNU. This contains the 3′ end region of ura4+ under the control of the nmt1+ promoter. Locations of the probes used in the TRO analysis are indicated. Selected mutant strains and a wild-type strain were transformed with pNU, and run-on analysis was performed according to Birse et al. (1997)
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Amino Acid Sequence Alignment between CstF-64, Rna15p, and Ctf1
Identical residues are boxed in black, and similar amino acids are boxed in gray. The N-terminal RNA binding domain is also indicated. The tyrosine at position 260 of Ctf1 is mutated into a stop codon in the mutant strain ctf1-70 as indicated
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Physical Interactions of Ctf1
(A) RNA interactions of Ctf1. Recombinant GST-Ctf1 was incubated with contiguous in vitro-transcribed RNA probes covering the four ura4+ 3′ end formation regions described in the diagram. The products of the crosslinking were treated with RNase and fractionated on a 10% SDS–PAGE, dried, and exposed to an autoradiographic film.
(B) Scheme of the interaction domains detected by the two-hybrid system. The N-terminal RNA binding domain (RBD) of Ctf1 is indicated by a striped box. The C-terminal domain (including the conserved domain, drawn in gray) was used as bait in a two-hybrid screen. A prey was obtained that contains the C-terminal domain of Res2. This C-terminal part interacts with Cdc10 (indicated as a checked box).
(C) GST pull-down experiments. Res2 was transcribed and translated in vitro in the presence of [35S]methionine. 35S-labeled Res2 was then incubated with GST alone or GST fused to either Res2 or Res1 followed by incubation with glutathione-Sepharose beads. After extensive washing, the attached proteins were eluted with SDS-loading buffer and fractionated on a 10% PAGE gel
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Effect of the Mutation of the S. pombe MBF Components in 3′ End Formation
(A) Run-on analysis. The pNU diagram is described in Figure 2. Run-on analysis of mutant strains is shown as indicated. Wild-type, ctf1-70, deletion strains of res2, res1, rep2, and a temperature-sensitive allele of cdc10 were transformed with plasmid pNU, and run-on analysis was performed as described. The cdc10ts strain was incubated at 37°C for 90 min prior to TRO analysis. Quantitation of this data is presented on the right-hand side. The M13 background signal was subtracted from each probe. The results were also normalized according to the U content of each probe, with the value for probe 1 fixed as 100.
(B) Steady-state analysis. Total RNA was extracted from strains growing in minimal medium without thiamine in order to activate nmt1 transcription. RNA was subjected to Northern blot analysis using a nmt1 StuI-XhoI probe. The filter was stripped and reprobed with a 18S rRNA probe (Hansen et al., 1998). The nmt1 signal was quantified, normalized against rRNA, and expressed relative to wild-type (set as 1)
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
Effect of the Mutation of S. cerevisiae MBF Components on 3′ End Formation
(A) Diagram depicting the GAL cluster. The location of the probes used in TRO are indicated underneath. The GAL10 and GAL7 transcripts are indicated by arrows, and the bicistronic transcript produced by mutation in poly(A) factors or signals is indicated by a dashed arrow.
(B) Northern blot analysis of the GAL genes from deletion mutants in the MBF subunits. Total RNA from deletion mutants in the MBF subunits and the wild-type strain W303-1a was separated on a 1.5% formaldehyde gel, blotted, and hybridized with a BglII-SalI probe from the GAL region that covers both transcripts. The filter was stripped and hybridized with a rRNA 18S probe (Hansen et al., 1998). Below each lane the GAL7/GAL10 ratio is indicated.
(C) TRO analysis from the wild-type strain W303-1a and the Δmbp1 strain. Probe A is a fragment of the actin gene, and M13 is a control phage without insert. Quantitation of this data is presented on the right-hand side. The M13 background signal was subtracted from each probe. The results were also normalized according the U content of each probe, with the value for probe 3 fixed as 100
Molecular Cell 2001 7, DOI: ( /S (01) )