Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

Zantema, A.; Maassen, J. Antonie; Kriek, Jan; Möller, W.

doi:10.1021/bi00256a006

Cited by 17 publications

(15 citation statements)

References 34 publications

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“…Mutants of ribosomal proteins containing single-Cys residues can be reacted with maleimide-conjugated dyes. Labeled and purified proteins are then incorporated into preassembled ribosomes lacking these specific proteins (7,8). A second strategy exploits the introduction of metastable hairpins into surface-exposed loops of rRNA (9).…”

Section: Resultsmentioning

confidence: 99%

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Fuchs

Petrov

Marceau

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.HCV IRES | translation initiation | human ribosomes | single-molecule FRET P rotein synthesis is a central process in health and disease (1, 2). The basic steps in translation have been mapped by genetic, biochemical, structural, and mechanistic studies. However, how translation is regulated and subverted, for example, during viral infection, remains poorly understood, especially in eukaryotes. All viruses compete for the cellular translation machinery to synthesize viral proteins required for virus proliferation. To that end, many viruses contain a structured internal ribosome entry site (IRES) in the 5′ untranslated region of their genome, which allows them to bypass the requirement for certain translation initiation factors. How IRESs achieve this goal remains unclear.Recently, it has been shown that structurally and evolutionarily very diverse IRESs, such as hepatitis C virus (HCV) IRES, cricket paralysis virus (CrPV) IRES (3), and others (4), require ribosomal protein eS25 (RPS25) for efficient translation initiation, indicating that RPS25/IRES interactions could be a universal feature of IRES-mediated translation. RPS25 is located on the back of the head of the 40S ribosomal subunit, distal to the mRNA entry channel but proximal to different IRES RNAs as shown by cryo-EM structures of 80S:CrPV IRES and 80S:HCV IRES complexes. RPS25 is not essential for cap-dependent translation, suggesting that IRES/RPS25 interactions are required to bypass the requirement for the full set of initiation factors (3).Translation initiation is a multistep process, with kinetics and dynamic interrogation refractory to conventional biochemical and biophysical methods. Single-molecule approaches provide insight into compositional and conformational dynamics of these asynchronous processes by following individual molecular events in real time. In bacteria, single-molecule methods allow direct obser...

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Section: Resultsmentioning

confidence: 99%

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Fuchs

Petrov

Marceau

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…On the other hand, under conditions which normally lead to the exclusive occupation of the so-called strong L7/L12 binding site on the 50S cores (Zantema et al, 1982a,b;Thielen et al, 1984), about 1.4 polypeptides of this double-labeled LI2 could be bound per 50S subunit. This value is to be compared with the 1.7 equiv of labeled L7/L12 which is normally bound (Zantema et al, 1982a;Thielen et al, 1984). Therefore, the energy transfer observed most likely was due to transfer between DACM and fluorescein attached to different polypeptides.…”

Section: Discussionmentioning

confidence: 99%

“…Labeling Procedures. The purified proteins L7 and LI2 were labeled as described before (Zantema et al, 1982a;Maassen et al, 1983). L7 was labeled with a hydrazine derivative of fluorescein or with DACM at the position of Lys-51, with a specificity of about 90%.…”

Section: Methodsmentioning

confidence: 99%

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Dimer structure of the ribosomal protein L7/L12 probed by energy transfer

Thielen¹,

Maassen²,

Kriek³

et al. 1984

Biochemistry

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“…After appropriate derivatization, distances within a dimer and between dimers marked by residues Met1, Lys29, and Lys51 of L12, and within the pentameric complex, using in addition Cys70 of L10, have been determined by FRET measurements and crosslinking [145][146][147] (Table 1). Comparison to the corresponding distances of the Cα-atoms in the crystal structures shows some disagreements with both crystalline dimer modes ( Table 1).…”

Section: An Effort To Reconcile L12 Crystal Structures With That In Smentioning

confidence: 99%

Structure and Function of the Acidic Ribosomal Stalk Proteins

Wahl

Möller²

2002

curr protein pept sci

106

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The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The ...

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Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

Cited by 17 publications

References 34 publications

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Dimer structure of the ribosomal protein L7/L12 probed by energy transfer

Structure and Function of the Acidic Ribosomal Stalk Proteins

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