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. 2012 Feb 29;40(12):5625–5636. doi: 10.1093/nar/gks200

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — The Tlc1 upstream terminator depends on the Nrd1/Nab3 transcription termination pathway. (A) Diagram of the ACT1–CUP1 fusion reporter gene with a cloned intronic terminator and the expected phenotypes on copper plates for recognition/read-through of the terminator. (B) Tenfold serial dilutions of cultures with cup1Δ strain 46a (WT), nrd1-5 (nrd1) or nab3-11 (nab3) and containing ACT1–CUP1 constructs with no terminator (No term), snoRNA terminator (SNR13 term) or Tlc1 terminator (TLC1–1233). A strain harboring no ACT1–CUP1 construct is used as a control for copper sensitivity (No CUP1). Cultures were spotted on medium lacking leucine and containing 0 or 0.5 mM added copper and incubated for 3 days at permissive (23°C) or semi-permissive (27°C for nab3, 32°C for nrd1) temperatures. (C) Same serial dilutions spot test as in (B) with strain 46a containing ACT1–CUP1 constructs with no terminator (No term), snoRNA terminator (SNR13 term) or Tlc1 terminator (TLC1–1233 term). The Tlc1 terminator is either unchanged (WT) or contains point mutations in the consensus Nrd1p binding site (Nrd1 mBS), in the consensus Nab3p binding site (Nab3 mBS) or in both sites (Nrd1/Nab3 mBS). A strain harboring no ACT1–CUP1 construct is used as a control for copper sensitivity (No CUP1). (D) Northern blot analysis of RNAs produced from ACT1–CUP1 reporter gene constructs using a CUP1-specific probe. TLC1–1233-25°C and TLC1–1233-32°C: RNAs derived from wild-type strain (46a) carrying the TLC1–1233 construct grown at 25°C or 32°C. nrd1-TLC1–1233-25°C and nrd1-TLC1–1233-32°C: RNAs derived from cells carrying the nrd1-5 ts allele and the TLC1–1233 construct grown at permissive (25°C) or semi-permissive (32°C) temperature. TLC1–1233-Nab3mBs: RNA derived from wild-type 46a cells carrying the TLC1–1233 construct with a point mutation in the Nab3p binding site. The TLC1–1209 construct is used as a negative control for transcription termination and the SNR13 snoRNA terminator is used as a positive control for termination (SNR13 term). A strain harbouring no ACT1–CUP1 construct is used as a negative control for hybridization (No CUP1). The RNA was hybridized to a probe specific to the CUP1 coding sequence downstream of the ACT1 intron. Size estimations in nucleotides indicated on the left were obtained after visualization of the 18S rRNA by methylene blue staining and after rehybridization with a terminally labelled oligonucleotide specific for the U1 snRNA (data not shown). The 18S rRNA visualized by methylene blue staining was used as a loading control.