Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli

Czworkowski, John; Odom, Obed W.; Hardesty, Boyd

doi:10.1021/bi00233a026

Cited by 44 publications

(28 citation statements)

References 29 publications

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“…Ribosomal protein-mapping studies indicate that E. coli S21 protein is in the platform region of the small subunit, the site of Shine-Dalgarno and codon-anticodon interactions (8). S21 is in very close proximity to both the 16S rRNA and the initiation region of mRNAs, as shown by cross-linking and resonance energy transfer experiments (15,21,48). However, the in vivo function of S21 has not been studied genetically.…”

Section: Discussionmentioning

confidence: 99%

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Green-Willms

Fox

Costanzo

1998

Molecular and Cellular Biology

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Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5 untranslated leaders (5-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5-UTLs which, in conjunction with mRNAspecific translational activation, is required for organellar translation initiation.While mitochondrial ribosomes exhibit distinct similarities to bacterial (eubacterial) ribosomes (30, 69), the yeast mitochondrial translation system has many intriguing differences from bacterial systems. Mitochondrial ribosomes have more proteins than do bacterial ribosomes (23, 43). Of the mitochondrial ribosomal proteins whose sequences are known, some are simple homologs of their bacterial counterparts, others have domains homologous to bacterial ribosomal proteins attached to domains with no recognizable homology to any known proteins, and still others are completely unrelated to bacterial ribosomal proteins (reviewed in reference 32). Saccharomyces cerevisiae mitochondrial mRNAs generally have long, AϩU-rich 5Ј untranslated leaders (5Ј-UTLs) lacking a typical Shine-Dalgarno sequence (11,27,31). While the mechanism of start site selection remains obscure in this system, translation initiation on most or all yeast mitochondrial mRNAs requires membrane-bound mRNA-specific activator proteins whose targets lie in the 5Ј-UTLs (reviewed in reference 27). These mRNA-specific activators appear to play a dual role in mitochondrial gene expression: tethering the synthesis of the very hydrophobic mitochondrial gene products to the inner membrane (27) and modulating the translation levels of individual mRNAs (63).We have focused on translation of the COX2 and COX3 mRNAs, which encode subunits II and III of cytochrome c oxidase, respectively. Previous studies have identified their mRNA-specific translation...

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Section: Discussionmentioning

confidence: 99%

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Green-Willms

Fox

Costanzo

1998

Molecular and Cellular Biology

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“…The reaction of the 5Ј-end with ethylenediamine or cystamine in the presence of a carbodiimide introduces a 5Ј-amino group that can be reacted with a fluorophore succinimidyl ester. Phosphorothioates can be introduced enzymatically at the 5Ј-or 3Ј-end using ATP␥S and polynucleotide kinase 18,19 or a 3Ј-phosphorothioate nucleotide and RNA ligase, 20 respectively, and reacted with haloacetyl derivatives of fluorescent dyes.…”

Section: Chemical and Enzymatic Methods For Labeling Of Dna And In VImentioning

confidence: 99%

Time-resolved fluorescence resonance energy transfer: A versatile tool for the analysis of nucleic acids

2002

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The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 A, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor-acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids.

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“…The second step was to label the 5′-terminal of P 5 with a perylene residue by using PeMIA as a precursor. As previously described (12), we did this labeling by the method of Czworkowski et al (17). The synthesized probe ( Fig.…”

Section: Perylene-labeled Pmentioning

confidence: 99%

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

Masuko

Ohuchi²,

Sode³

et al. 2000

Nucleic Acids Research

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We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32mer and its complementary two sequential 16mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of~100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 Å (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleotides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2

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Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli

Cited by 44 publications

References 29 publications

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Time-resolved fluorescence resonance energy transfer: A versatile tool for the analysis of nucleic acids

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

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