1. The Zipper Model of Translational Control
Ibrahim Yaman, James Fernandez, Haiyan Liu, Mark Caprara, Anton A. Komar, Antonis E. Koromilas, Lingyin Zhou, Martin D. Snider, Donalyn Scheuner, Randal J. Kaufman, Maria Hatzoglou 
Cell 
Volume 113, Issue 4, Pages (May 2003)
DOI: /S (03)

2. Figure 1
Induction of Cat-1 IRES Activity Depends on Translation of the uORF and Involves Long-Range RNA Interactions
C6 cells were transiently transfected with bicistronic expression vectors containing the cat1-5′UTR DNAs shown in (A). Numbers indicate the position of the nucleotides relative to the cat-1 ORF A+1TG. Exons 1–3 are shown in blue, red, and yellow, respectively. (B) Following transfection (36 hr), cells were shifted to fed (F) or starved (S) conditions for 9 hr. Cell extracts were prepared and LUC and CAT activities were measured. The LUC/CAT ratio relative to the cat1(−270) value in fed cells is shown. The numbers indicate the constructs used. The bars represent the mean ± SEM of three independent experiments. For construct one, the increased LUC/CAT ratio caused by starvation is due to a 20% decrease in CAT and a 4.4-fold increase in LUC. For all the constructs, starvation caused a 10%–30% decrease in CAT activity.
Cell  , DOI: ( /S (03) )

3. Figure 2
Induction of IRES-Mediated Translation Is Independent of the uORF Peptide Sequence
C6 cells were transiently transfected with bicistronic expression vectors containing cat1-5′UTR sequences in the intercistronic region (A). Construct one is the wild-type cat1(−270). Constructs two and three have the indicated mutations. (B) Alignment of the peptides encoded by the uORFs in constructs one and two. (C) Following transfection (36 hr), cells were shifted to fed (F) or starved (S) conditions for 9 hr and the LUC/CAT ratios were determined as described in Figure 1.
Cell  , DOI: ( /S (03) )

4. Figure 3
The 5′ End of the Cat-1 uORF Is Inhibitory to Ribosome Movement
(A) RNAs containing the indicated sequences from the cat-1 mRNA leader and 610 nt from the 5′ end of the LUC ORF were prepared as described in Experimental Procedures. All RNAs contained the nucleotide G at the 5′ end.
(B) RNAs were translated in vitro in the presence of [35S]Met and labeled products were analyzed on SDS-PAGE gels. Radioactivity was analyzed by phosphorimaging. Numbers indicate the vectors used to generate RNAs. The relative amount of radioactivity is shown below the autoradiograms. The bars indicate standard errors of scanning of the bands.
(C) In vitro transcribed full-length LUC RNAs containing the cat1(−270), cat1(−270)mut, and cat1(−192) leaders were cotranslated in vitro with a CAT RNA as an internal control. LUC and CAT activities were measured, and the LUC/CAT ratio is given relative to the cat1(−270) value.
Cell  , DOI: ( /S (03) )

5. Figure 4
The Inducible Cat-1 IRES Is Contained within the 192 Nucleotides at the 3′ End of the Leader
(A–C) C6 cells were transiently transfected with bicistronic expression vectors containing the cat1-5′UTR sequences shown between the CAT and LUC cistrons. Mutations in the cat1-5′UTR sequences are underlined. The LUC/CAT ratios in fed and starved cells were determined as described in Figure 1.
(D) Predicted structure of cat1(−192) using mfold software. Blue lines indicate nucleotides absent from the mouse sequence. Blue nucleotides indicate substitutions in the mouse sequence.
(E) MEFs S/S and A/A cells were transfected with bicistronic vectors containing the cat1(−270) or cat1(−192) leaders between the CAT and LUC cistrons (Figure 1A, construct one, and Figure 4A, construct one). Cell treatments and analysis of the data were as described in (B).
Cell  , DOI: ( /S (03) )

6. Figure 5
Translation of the uORF Mediates Dynamic RNA Interactions within the Cat-1 Leader
C6 cells were transiently transfected with bicistronic expression vectors containing the cat1-5′UTR sequences shown in (A) between the CAT and LUC cistrons. Mutations within the cat1-5′UTR sequences are underlined. The LUC/CAT ratios in fed and starved cells were determined as described in Figure 1. The numbers at the bottom of (B) and (C) indicate the constructs that were used.
Cell  , DOI: ( /S (03) )

7. Figure 6
Nuclease and Chemical Sensitivity Mapping of the Cat1(−270) and Cat1(−192) Leaders
In vitro transcribed cat1(−270) (A) and cat1(−192) (B) RNAs were 5′ end labeled, gel purified, renatured in the presence or absence of Mg2+, and incubated with the nucleases (T1 and V1) or DEPC/aniline (see Experimental Procedures). The cleavage products were resolved on 7% acrylamide/7 M urea sequencing gels. The radioactivity on the gels was measured by phosphorimaging. Numbers on the left indicate the positions of residues relative to the cat-1 ORF ATG. RNAs subjected to alkaline hydrolysis and T1 nuclease are shown on the left. The tables show a summary of selected cleavages from the experiments in (A) and (B). The tables also list cleavages within the region −1 and −20, which were studied using chemical and enzymatic probing of 3′ end-labeled RNAs (data not shown).
Cell  , DOI: ( /S (03) )

8. Figure 7
Inhibition of Translation by the Cat-1 Leader in Cell-Free Assays Involves Long-Range RNA Interactions
(A) Proposed conformations of the cat-1 mRNA leader. In the absence of uORF translation, the RNA is involved in a structure that inhibits formation of the inducible IRES (left). The structure shown is a working model that is consistent with our data. When the uORF is translated, the structured cat-1 leader unfolds, allowing new RNA interactions within the 192 nt at the 3′ end (right). The RNA structure is the IRES that can be induced by amino acid starvation. Cleavage sites as determined in Figure 6 are shown.
(B–E) RNAs containing the indicated cat-1 sequences attached to a truncated LUC ORF were translated in vitro in the presence of [35S]Met and analyzed as in Figure 3. The numbers indicate the vectors in (A) used to generate the RNAs.
(F) The zipper model of translational control. This kinetic model involves three stages. In stage I, which occurs in the absence of uORF translation, the IRES is in an unproductive conformation and its activity cannot be induced by amino acid starvation. In stage II, which is formed upon translation of the uORF, the IRES is in a productive conformation that can be induced by amino acid starvation. In stage III, which occurs during amino acid starvation, an ITAF binds the inducible IRES converting it to the induced state, resulting in the initiation of translation at the cat-1 ORF. The arrows indicate the favored kinetic states.
Cell  , DOI: ( /S (03) )