1. Involvement of 16S rRNA Nucleotides G1338 and A1339 in Discrimination of Initiator tRNA 
Laura Lancaster, Harry F. Noller 
Molecular Cell 
Volume 20, Issue 4, Pages (November 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Insertion of the MS2 Coat Protein RNA Binding Sequence into 16S rRNA for Affinity Purification of Mutant 30S Subunits and Analysis of Their Purity
(A) 16S rRNA with helix 6 (the spur) boxed and shown in detail (B).
(C) Engineered construct showing the insertion of the MS2 affinity tag (bold) into helix 6.
(D) Primer extension analysis of rRNA extracted from ribosomes before (A) and after (B) affinity purification. The proportion of plasmid-encoded 16S rRNA (containing the MS2 affinity tag sequence) is given below each lane.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Expression of 16S rRNA Genes with Mutations at G1338 and A1339 in Addition to the Inserted MS2 Affinity Tag Sequence
E. coli strains containing plasmid pLK35.MS2, which encodes MS2-tagged 30S subunits from a temperature-inducible promoter (indicated as “WT”), or with additional 16S rRNA mutations as indicated were grown at 30°C (repressive temperature) to saturation. Twenty microliters of 10−4, 10−5, 10−6, and 10−7 dilutions was spotted and incubated at 30°C or 42°C (derepressive temperature) for 24 hr.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
In Vitro Translational Activity of Ribosomes Containing 30S Subunits with 16S rRNA Mutations at G1338 and A1339 and Comparison between Their Activities In Vivo and In Vitro
(A) Poly U-directed [14C]-Phe incorporation into peptides was measured at the indicated time points. Results were normalized to the maximum Phe incorporated (approximately 30 pmol per pmol of ribosome) by wild-type subunits. The results from at least two independent experiments were plotted and fit to a single exponential curve. Symbols are as follows: wild-type, □; G1338A, ■; G1338C, ♦; G1338U, ▵; A1339C, ▴; A1339G, ○; G1338A:A1339G, ●.
(B) In vivo β-galactosidase production (Table 1) from 30S subunits without (black bar) or with (dotted bar) the MS2 affinity tag and their observed rates of in vitro poly-Phe synthesis (grey bar). All are normalized to wild-type subunit activity.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Analysis of tRNA Binding to 30S Subunits with Mutations at G1338 and A1339
(A) Anticodon stem-loop sequences (Sprinzl et al., 1998) of the tRNAs used for in vitro experiments.
(B) Sequence of the mRNA with the corresponding P site codons indicated above.
(C) Toeprinting analysis of 30S subunit and (D) 70S ribosome binding to different tRNAs.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Initiation Factor IF2 Stimulation of fMet-tRNAfMet Binding to Mutant 30S Subunits
Nitrocellulose filter binding of complexes containing 30S subunits, mRNA, [35S]-fMet-tRNAfMet, and increasing amounts of IF2 (see Experimental Procedures). Results were normalized to the highest counts obtained by wild-type 30S subunits, which correspond to about 0.8 pmols of fMet-tRNAfMet bound per pmol of 30S. Symbols represent the following: wild-type, □; G1338A, ■; G1338C, ♦; G1338U, ▵; A1339C, ▴; A1339G, ○; G1338A:A1339G, ●.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Initiation Factor IF3-Dependent Displacement of Elongator tRNAs with fMet-tRNAfMet in Mutant 30S Subunits
First, complexes were formed containing 30S subunits, mRNA, and tRNAPhe (A), tRNACys (B), tRNAMet (C), or tRNAyeast Phe (D) and then [35S]-fMet-tRNAfMet was added along with increasing amounts of IF3 (see Experimental Procedures). IF3-mediated exchange of [35S]-fMet-tRNAfMet for each elongator tRNA was monitored by nitrocellulose filter binding and was normalized to the highest counts obtained by wild-type 30S subunits, which correspond to about 0.6 pmol fMet-tRNAfMet bound per pmol of 30S. Symbols represent the following: wild-type, □; G1338A, ■; G1338C,♦; G1338U, ▵; A1339C, ▴; A1339G, ○; G1338A:A1339G, ●.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
16S rRNA Bases G1338 and A1339 Form Type II and Type I Minor Interactions
16S rRNA Bases G1338 and A1339 form (A) Type II and (B) Type I minor interactions (Nissen et al., 2001) with consecutive G-C pairs of a tRNA analog in the P site of the 30S subunit crystal structure (Carter et al., 2000).
Molecular Cell  , DOI: ( /j.molcel )
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