RESUMO
Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.
Assuntos
Escherichia coli/genética , Impressão Molecular/métodos , Pseudouridina/química , RNA Ribossômico 16S/química , RNA Ribossômico 23S/química , Compostos de Anilina/química , Antibacterianos/farmacologia , Fracionamento Celular/métodos , DNA Complementar/biossíntese , DNA Complementar/química , DNA Complementar/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gentamicinas/farmacologia , Hidroliases/genética , Hidroliases/metabolismo , Ligantes , Cloreto de Magnésio/farmacologia , Neomicina/farmacologia , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Pseudouridina/genética , Pseudouridina/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Transcrição Reversa , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/efeitos dos fármacos , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Ribossomos/metabolismo , Ésteres do Ácido Sulfúrico/químicaRESUMO
BACKGROUND: Healing of burns is a complex process and very few effective treatments exist to facilitate the burn recovery process. Human acidic fibroblast growth factor 1 (FGF-1) plays an important role in a variety of biological processes, including angiogenesis, and tissue repair. Salvia miltiorrhiza is widely used in traditional Chinese medicine as an herb for the treatment of various diseases, including cardiovascular and cerebrovascular diseases, and traumatic injuries. We present that expression of FGF-1 in S. miltiorrhiza significantly accelerates the healing of burn wounds. RESULTS: The human fgf-1 gene was fused with a barley α-amylase signal peptide DNA sequence and driven by a 35S promoter for constitutive expression in transgenic S. miltiorrhiza plants. The highest yield of recombinant FGF-1 obtained from leaves of transgenic S. miltiorrhiza lines was 272 ng/fresh weight. Aqueous extracts from transgenic S. miltiorrhiza exhibited FGF-1 activity approximately 19.2-fold greater than that of the standard FGF-1. Compared to the standard FGF-1 or the extracts obtained from non-transgenic plants, it stimulated proliferation of Balb/c 3 T3 mouse fibroblast cells assessed with the standard MTT assay and promoted angiogenesis in the chicken embryo chorioallantoic membrane (CAM) assay. Topical application of the extract significantly accelerated the burn wound healing process. CONCLUSIONS: The product appears to retain the biological activity of both FGF-1 as well as the medicinal properties of the plant. The extracts from transgenic S. miltiorrhiza combines the therapeutic functions of FGF-1 and the medicinal plant, S. miltiorrhiza. Topical application of the product can reduce the costs associated with extraction, purification, and recovery.