The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry

BACKGROUND: Onychomycosis has become one of the common fungal infection. However, highly reliable and sensitive methods of detecting and identifying causative fungi of onychomycosis are not established yet. Polymerase chain reaction (PCR) analysis of clinical specimens including blood, sputum, urine, and cerebrospinal fluid collected from patient systemically infected fungus is known as a sensitive diagnostic method. But it has been questionable whether PCR analysis is also applicable to onychomycosis. OBJECTIVE: The purpose of this study was to develop a DNA-based diagnostic method to improve the sensitivity and specificity of detection and identification of pathogenic fungi of onychomycosis. METHODS: To detect the fungi in the nail, PCR was performed by using 4 sets of primer (TR1 & TR2, NS5 & NS6, B2F & B4R and CA1 & CA2) designed in conserved sequences of the small ribosomal subunit (185-rRNA) genes and restriction enzyme analysis of amplified product by Hae III was done to identify species. Nail specimens were obtained from 19 cases of onychomycosis confirm by fungus culture. RESULTS: 1. Preparation of nail powder, which is necessary for removal of keratin, and composition of lysis buffer with guanidinium thiocyanate, Tris-HCl, and beta -mercaptoethanol are the most proper modalities for isolation of fungal DNA from fungus-infesting nails. 2. Specific fragments of the 18S-rRNA gene of fungi, 581 bp, 308 bp, 688 bp and 1106 bp were amplified respectively. From sequences of 18S-rRNA gene of fungi by universal primers, dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes) and yeast (Candida albicans) yielded identical products. 3. Using Hae III endonuclease, digested patterns of fragment of Trichophyton rubrum and Candida albicans resulted in different pattern. CONCLUSION: This method released enough DNA from fungus-infected nails to result in proper amplification and it can be possible to differentiate dermatophytes, yeasts, and molds using Hae III endonuclease. The present study is the first one to demonstrate the feasibility of this molecular biologic approach to identify fungi in the infected nail. Therefore, precise detection and identification of the causative fungi would be of help in investigating distribution of the causative fungi of onychomycosis as well as appropriate treatment of the disease.
Arthrodermataceae ; Candida albicans ; Cerebrospinal Fluid ; Conserved Sequence ; Diagnosis* ; DNA ; DNA, Fungal ; Fungi* ; Guanidine ; Humans ; Onychomycosis* ; Polymerase Chain Reaction* ; Restriction Mapping* ; Ribosome Subunits, Small ; Sensitivity and Specificity ; Sputum ; Trichophyton ; Yeasts