ABSTRACT
Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5' untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.
Subject(s)
Bacterial Proteins/metabolism , Nucleic Acid Conformation , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Ribosomal Protein S6/metabolism , Ribosomal Proteins/metabolism , 5' Untranslated Regions/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Pairing , Base Sequence , Binding Sites , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Models, Molecular , Molecular Sequence Data , Operon/genetics , Protein Binding , Protein Structure, Tertiary , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , Ribosomal Protein S6/chemistry , Ribosomal Protein S6/genetics , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomes/chemistry , Ribosomes/genetics , Ribosomes/metabolism , Sequence Homology, Nucleic Acid , Thermus thermophilus/genetics , Thermus thermophilus/metabolismABSTRACT
PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.
