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Comparison of mRNA binding by Met-tRNAf binding protein and mRNA-associated proteins
Abstract:
One of the heterogeneous mRNA binding activities in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes co-purified to apparent homogeneity through phosphocellulose and DEAE-cellulose chromatography and isoelectric focusing with the GTP-dependent Met-tRNAf binding protein. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following iodination revealed putative subunits of 51,000 and 39,000 apparent molecular weights. Specificity of mRNA binding by this protein was suggested since the ability of poly(A)-rich mRNA to compete for binding of [3H]poly(A)-rich mRNA exceeded by 10- to 100-fold that of most natural or synthetic RNAs tested, except for the hybrid poly(G) - poly(C) which was almost as effective, and poly(G), which was more effective, at competing for protein-dependent binding. The mRNA binding activity exhibited complete GTP independence and no apparent divalent cation requirement. GDP inhibited Met-tRNAf binding but neither GDP, GMP, nor 7-methylguanosine 5'-monophosphate inhibited mRNA binding by this protein. Similar data were obtained with respect to the ability of natural or synthetic RNAs to compete for binding of [3H]poly(A)-rich mRNA by proteins associated with purified rabbit reticulocyte polyribosomal mRNA-protein particles; while poly(A) was an ineffective competitor, poly(G) was more effective than even mRNA at competing for protein-dependent binding. No significant binding of Met-tRNAf by mRNA-protein particles was detected. Polyacrylamide gel electrophoresis following reduction of mRNA-protein particles revealed apparent co-migration of a major protein with one subunit of the GTP-dependent Met-tRNAf binding protein, but no protein comparable to the 39,000 dalton subunit protein.
Insights
Researchers identified a novel mRNA binding protein in rabbit reticulocytes. This protein, distinct from the GTP-dependent Met-tRNAf binding protein, shows specific affinity for mRNA, particularly poly(G) sequences.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- RNA-Protein Interactions
Background:
- Rabbit reticulocyte polyribosomes contain heterogeneous mRNA binding activities.
- A GTP-dependent Methionine tRNA (Met-tRNAf) binding protein is known to be involved in translation initiation.
- The precise nature and specificity of other mRNA binding proteins in this system remain to be fully elucidated.
Purpose of the Study:
- To isolate and characterize a specific mRNA binding protein from rabbit reticulocyte polyribosomes.
- To investigate the binding characteristics and specificity of this isolated protein.
- To determine its relationship, if any, with the known GTP-dependent Met-tRNAf binding protein.
Main Methods:
- Purification of mRNA binding activity using phosphocellulose and DEAE-cellulose chromatography, and isoelectric focusing.
- Analysis of protein subunits using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after iodination.
- Competition assays using radiolabeled mRNA ([3H]poly(A)-rich mRNA) and various natural or synthetic RNAs to assess binding specificity.
- Investigation of GTP and divalent cation requirements for mRNA binding.
- Analysis of proteins associated with purified mRNA-protein particles using SDS-PAGE.
Main Results:
- A novel mRNA binding activity was purified and found to co-purify with the GTP-dependent Met-tRNAf binding protein.
- The purified protein exhibited apparent subunits of 51,000 and 39,000 molecular weights.
- The protein displayed specific mRNA binding, with poly(A)-rich mRNA being a strong competitor, and poly(G) and poly(G)-poly(C) being even more effective competitors.
- mRNA binding was independent of GTP and divalent cations.
- GDP inhibited Met-tRNAf binding but not mRNA binding.
- Purified mRNA-protein particles showed preferential binding of poly(G) over mRNA, and no significant Met-tRNAf binding was detected.
Conclusions:
- A distinct mRNA binding protein, separable from the Met-tRNAf binding activity, was identified and characterized.
- This protein demonstrates a unique binding preference for specific RNA sequences, notably poly(G).
- The findings suggest the presence of multiple, distinct RNA-binding proteins involved in translational regulation within rabbit reticulocytes.