Journal: Nucleic Acids Research

Article Title: A new RNA–DNA interaction required for integration of group II intron retrotransposons into DNA targets

doi: 10.1093/nar/gkab1031

Figure Lengend Snippet: Secondary structure of the Escherichia coli subgroup IIB1 intron EcI5 and its DNA target site. ( A ) The secondary structure of the EcI5 intron is coloured by major domains (I to VI). Tertiary base-pairing interactions between the intron and its 5′ and 3′ flanking exons are designated as EBS (Exon Binding Site)—IBS (Intron Binding Site) pairings whereas long-range interactions involving intronic sequences are designated by greek letters. The EBS2a and IBS2a sites studied in this work are highlighted in red. For mobility assays, most of the RT ORF in domain IV (coloured in black) was deleted and replaced by a T7 RNA polymerase promoter (PT7) as indicated by the double-headed arrows. The portion of domain IV coloured in red corresponds to the sequences still present in the EcI5 ‘ribozyme’ construct used for mobility experiments (see Materials and Methods and Figure ). The IVa subdomain contains the high affinity binding site for the EcI5 RT enzyme. The translation signals used for synthesis of the RT in the natural intron are circled in green. SD: Shine-Dalgarno sequence. ( B ) Natural DNA recognition site of the EcI5 intron. Coloured nucleotides on the top strand (C-18, C-17, A-15, A-14 and T + 5) are those previously identified as being critical for EcI5 mobility and suggested to be directly recognized by the RT . The present work demonstrates that DNA position A-14 (in red), now named IBS2a, is in fact engaged in a Watson–Crick pairing with the intron EBS2a site highlighted in red (see text). TS: Target site.

Article Snippet: E. coli HMS174(DE3) strain (Novagen) was used for all intron mobility experiments.

Techniques: Binding Assay, Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: A new RNA–DNA interaction required for integration of group II intron retrotransposons into DNA targets

doi: 10.1093/nar/gkab1031

Figure Lengend Snippet: Impact of the identity of the EBS2a–IBS2a pairing on mobility efficiency. The identity of the bases at the EBS2a and IBS2a sites is in upper and lower case, respectively. Appropriate donor and recipient plasmids were co-transformed into E. coli HMS174(DE3) and intron mobility was triggered with 100 μM IPTG at 37°C for 1 h. Mobility efficiencies were calculated as the ratio of (Amp R + Tet R )/(Amp R + Cam R ) colonies (see Materials and Methods). Graph bars are the mean value from at least five mobility efficiency values. Error bars represent the standard error of the mean.

Article Snippet: E. coli HMS174(DE3) strain (Novagen) was used for all intron mobility experiments.

Techniques: Transformation Assay

Journal: Nucleic Acids Research

Article Title: A new RNA–DNA interaction required for integration of group II intron retrotransposons into DNA targets

doi: 10.1093/nar/gkab1031

Figure Lengend Snippet: Impact of the EBS2a–IBS2a interaction on intron splicing in vivo . Primer extension reactions were performed on total cellular RNA extracted from E. coli HMS174(DE3) cells expressing EcI5 ribozyme (intronΔORF) precursors with the indicated matched or mismatched EBS2a–IBS2a combinations (in red; see also Materials and Methods). One representative 5% acrylamide-urea primer extension gel is shown. L(lariat) and P(precursor) are migration markers generated by primer extension of in vitro transcribed RNA. The identity of the lariat ‘stop’ band was further confirmed with additional gels (not shown) that include a sequence generated from direct sequencing of an in vitro transcribed wt EcI5 intronΔORF precursor. The weak band between the ‘L’ and ‘P’ cDNAs was consistently observed in all experiments but was not used for quantification because its origin is unknown.

Article Snippet: E. coli HMS174(DE3) strain (Novagen) was used for all intron mobility experiments.

Techniques: In Vivo, Expressing, Migration, Generated, In Vitro, Sequencing

Journal: Protein Science : A Publication of the Protein Society

Article Title: Kinetic versus thermodynamic control of mutational effects on protein homeostasis: A perspective from computational modeling and experiment

doi: 10.1002/pro.3639

Figure Lengend Snippet: Parameters and in vivo protein folding fates of the clusters that make up our coarse‐grained proteome. The top four plots show the values of k f,i, k u,i, ΔG f,i, and k agg,i. Note the logarithmic scales for k f,i, k u,i, and k agg,I and the inverted scale for ΔG f,i. The cell division rate (λ) and mutation response categories are indicated in the plot for k u,i (see text). Data points are also colored by mutation response category: blue for Category 1, red for Category 2, green for Category 3, and gray for Category 4. Below the plots are qualitative indications of the responsiveness toward chaperoning by DnaK/DnaJ/GrpE and GroEL/GroES systems (++: excellent substrate; +: good substrate; −: poor substrate), the mutation response category, and the fractional proteomic abundances of each cluster. The bottom bar chart shows the fractional occupancy for each cluster of the natively folded (Ni), unfolded (Ui), aggregated (Ai + K:Ai), chaperone bound (K:Ui + G:Ui + G:Ni + D:Ui), and degraded states for E. coli under fast growth conditions (λ = 0.00053 s−1, corresponding to a doubling time of about 20 min).

Article Snippet: Escherichia coli strains and plasmids The E. coli strain K12 HMS174(DE3) (Novagen) was used as the background strain in all the experiments.

Techniques: In Vivo, Mutagenesis

Journal: Protein Science : A Publication of the Protein Society

Article Title: Kinetic versus thermodynamic control of mutational effects on protein homeostasis: A perspective from computational modeling and experiment

doi: 10.1002/pro.3639

Figure Lengend Snippet: Time courses of expression of MmCRABP1 variants in E. coli. Red lines and data points: total MmCRABP1 concentrations per cell. Blue lines and data points: soluble MmCRABP1 concentrations per cell. The difference between the total and soluble protein concentrations is the aggregated protein concentration (red shaded area). The fractions of soluble protein at the 3 h time point for all variants are indicated on the plots. Error bars indicate the standard errors of the mean.

Article Snippet: Escherichia coli strains and plasmids The E. coli strain K12 HMS174(DE3) (Novagen) was used as the background strain in all the experiments.

Techniques: Expressing, Protein Concentration