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Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model.

ABSTRACT: A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis. One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA. This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations. This interpretation was supported by evidence from both genetics and structural biochemistry. However, recently published X-ray crystallographic structures of the Thermus thermophilus 30S subunit at different stages of tRNA selection have raised doubts regarding the validity of this model. We have therefore revisited the model genetically by constructing a H27 quadruple mutation (C912G, C910G, G885C, and G887C), which would create multiple mismatches in the proposed alternative conformation without perturbing the native H27 conformation seen in the crystal structures. Inconsistent with the H27 switch model, cells containing pure populations of quadruple mutant ribosomes grow at essentially wild-type rates. The mutants used to construct the H27 switch model all carried A2058G in 23S rRNA and C1192U in 16S rRNA as selectable markers. The quadruple mutant carrying these additional marker mutations is deleterious, and we conclude that they have a synergistic effect when combined with other mutations and are not phenotypically silent. Their presence confounded the interpretation of the original mutant phenotypes and, in light of the viability of the quadruple mutant, we conclude that the genetic evidence no longer supports the model.

SUBMITTER: Rodriguez-Correa D

PROVIDER: S-EPMC1370515 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model.

Rodriguez-Correa Daniel D Dahlberg Albert E AE

RNA (New York, N.Y.) 20040101 1

A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis. One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA. This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations. This interpretation was supported by evidence from both ge ...[more]

PMID: 14681582

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Project description:RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.

Project description:BackgroundIxodes scapularis is a vector of several human pathogens in the United States, and there is geographical variation in the relative number of persons infected with these pathogens. Geographically isolated populations of I. scapularis have established or are in the process of establishing in southern Canada. Knowledge of the genetic variation within and among these populations may provide insight into their geographical origins in the United States and the potential risk of exposure of Canadians to the different pathogens carried by I. scapularis.MethodsPart of the mitochondrial (mt) 16S ribosomal (r) RNA gene was amplified by PCR from 582 ticks collected from southern Canada, and Minnesota and Rhode Island in the United States. Sequence variation was examined in relation to the predicted secondary structure of the gene. Genetic diversity among populations was also determined.ResultsDNA sequence analyses revealed 52 haplotypes. Most mutational alterations in DNA sequence occurred at unpaired sites or represented partial compensatory base pair changes that maintained the stability of the secondary structure. Significant genetic variation was detected within and among populations in different geographical regions. A greater proportion of the haplotypes of I. scapularis from the Canadian Prairie Provinces were found in the Midwest of the United States than in other regions, whereas more of the haplotypes of I. scapularis from the Canadian Central and Atlantic Provinces occurred in the Northeast of the United States. Nonetheless, 58% of I. scapularis were of a haplotype that occurs in the Midwest and Northeast of the United States; thus, their geographical origins could not be determined.ConclusionsThere is considerable genetic variation in the mt 16S rRNA gene of I. scapularis. There is some evidence to support the hypothesis that some lineages of I. scapularis in the Atlantic and Central Provinces of Canada may be derived from colonizing individuals originating in the Northeast of the United States, whereas those in the Prairie Provinces may be derived from individuals originating in the Midwest of the United States. However, additional genetic markers are needed to test hypotheses concerning the geographical origins of I. scapularis in Canada.

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Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model.

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Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model.

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