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Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

Williams SG, Hall KB - Biochemistry (2014)

Bottom Line: We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity.In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding.The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School , St. Louis, Missouri 63110, United States.

ABSTRACT
The U1A and U2B″ proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B″ to their respective snRNP is a well-known phenomenon, binding of U2B″ to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B″/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluorescence and isothermal titration calorimetry binding experiments. We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity. In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding. The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

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Schematicsof binding model and thermodynamic cycles for ternarycomplex formation. R, U, and M represent the RNA, U2A′, andRRM-containing protein (U1A or U2B″), respectively. αis the linkage parameter. KR and KU are the bimolecular binding constants forthe RRM–RNA and RRM–U2A′ interactions, respectively.The enthalpies associated with protein–protein and protein–RNAbinding are indicated by ΔHU andΔHR, respectively, and the enthalpyassociated with cooperativity between the two binding events is indicatedby Δh.
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fig2: Schematicsof binding model and thermodynamic cycles for ternarycomplex formation. R, U, and M represent the RNA, U2A′, andRRM-containing protein (U1A or U2B″), respectively. αis the linkage parameter. KR and KU are the bimolecular binding constants forthe RRM–RNA and RRM–U2A′ interactions, respectively.The enthalpies associated with protein–protein and protein–RNAbinding are indicated by ΔHU andΔHR, respectively, and the enthalpyassociated with cooperativity between the two binding events is indicatedby Δh.

Mentions: U1A or U2B″/U2A′titrations were performed in 250 mM KCl, 10 mM potassium phosphate(pH 8), 1 mM MgCl2, 40 μg/mL BSA, 5 mM DTT, and RNasin.Titrations were performed at 23 °C, with constant stirring. Fora single titration of U1A/U2B″ or U1A/U2B″/U2A′into fluorescein-labeled RNA, the cuvette and titrant concentrationof fluorescein-labeled RNA was held constant at 0.1 or 0.5 nM (thelower concentration was used for the highest-affinity interactions).The cuvette and titrant also contained identical concentrations ofU2A′. The sample was excited at 490 nm, and the emission intensityat 520 nm was recorded (excitation and emission slit openings of 8and 16 nm, respectively). U1A or U2B″ with or without U2A′was titrated into the RNA, and the fluorescence emission intensitywas recorded for each addition of protein. The intensity data wereconverted to fluorescence enhancement and normalized to the maximalfluorescence enhancement to represent the fraction of bound RNA. Titrationswere collected at multiple concentrations of U2A′, and thedata were globally fit in Scientist (Micromath) to eqs 1–4:1234where FM+UM isthe fraction of the total RNA, bound either to U1A/U2B″ (M)or to U2A′:U1A/U2B″ (UM); RT, UT, and MT are the total RNA, U2A′, and U1A/U2B″ concentrations,respectively; R, U, and M are the concentrations of free RNA, U2A′, and U1Aor U2B″, respectively; α is the cooperativity parameter;and KR and KU are the bimolecular association constants for the SNF–RNAand SNF–U2A′ interactions, respectively. The schematicfor data analysis in terms of a thermodynamic cycle of protein andRNA binding is illustrated in Figure 2.


Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

Williams SG, Hall KB - Biochemistry (2014)

Schematicsof binding model and thermodynamic cycles for ternarycomplex formation. R, U, and M represent the RNA, U2A′, andRRM-containing protein (U1A or U2B″), respectively. αis the linkage parameter. KR and KU are the bimolecular binding constants forthe RRM–RNA and RRM–U2A′ interactions, respectively.The enthalpies associated with protein–protein and protein–RNAbinding are indicated by ΔHU andΔHR, respectively, and the enthalpyassociated with cooperativity between the two binding events is indicatedby Δh.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4067145&req=5

fig2: Schematicsof binding model and thermodynamic cycles for ternarycomplex formation. R, U, and M represent the RNA, U2A′, andRRM-containing protein (U1A or U2B″), respectively. αis the linkage parameter. KR and KU are the bimolecular binding constants forthe RRM–RNA and RRM–U2A′ interactions, respectively.The enthalpies associated with protein–protein and protein–RNAbinding are indicated by ΔHU andΔHR, respectively, and the enthalpyassociated with cooperativity between the two binding events is indicatedby Δh.
Mentions: U1A or U2B″/U2A′titrations were performed in 250 mM KCl, 10 mM potassium phosphate(pH 8), 1 mM MgCl2, 40 μg/mL BSA, 5 mM DTT, and RNasin.Titrations were performed at 23 °C, with constant stirring. Fora single titration of U1A/U2B″ or U1A/U2B″/U2A′into fluorescein-labeled RNA, the cuvette and titrant concentrationof fluorescein-labeled RNA was held constant at 0.1 or 0.5 nM (thelower concentration was used for the highest-affinity interactions).The cuvette and titrant also contained identical concentrations ofU2A′. The sample was excited at 490 nm, and the emission intensityat 520 nm was recorded (excitation and emission slit openings of 8and 16 nm, respectively). U1A or U2B″ with or without U2A′was titrated into the RNA, and the fluorescence emission intensitywas recorded for each addition of protein. The intensity data wereconverted to fluorescence enhancement and normalized to the maximalfluorescence enhancement to represent the fraction of bound RNA. Titrationswere collected at multiple concentrations of U2A′, and thedata were globally fit in Scientist (Micromath) to eqs 1–4:1234where FM+UM isthe fraction of the total RNA, bound either to U1A/U2B″ (M)or to U2A′:U1A/U2B″ (UM); RT, UT, and MT are the total RNA, U2A′, and U1A/U2B″ concentrations,respectively; R, U, and M are the concentrations of free RNA, U2A′, and U1Aor U2B″, respectively; α is the cooperativity parameter;and KR and KU are the bimolecular association constants for the SNF–RNAand SNF–U2A′ interactions, respectively. The schematicfor data analysis in terms of a thermodynamic cycle of protein andRNA binding is illustrated in Figure 2.

Bottom Line: We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity.In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding.The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School , St. Louis, Missouri 63110, United States.

ABSTRACT
The U1A and U2B″ proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B″ to their respective snRNP is a well-known phenomenon, binding of U2B″ to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B″/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluorescence and isothermal titration calorimetry binding experiments. We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity. In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding. The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

Show MeSH