Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes

Holmberg, Lovisa; Melander, Yvette; Nygård, Odd

doi:10.1093/nar/22.14.2776

Cited by 22 publications

(22 citation statements)

References 43 publications

(79 reference statements)

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“…Thus, it seems reasonable to assume that the homologous helices have the same location in the 30 S and 40 S particles. If so, the affected sites in the central domain would be located at the protuberance side of the 40 S subunit close to the rRNA sites affected by subunit-subunit interaction (11), while the affected sites in the 5Ј-and 3Ј-domains would be positioned in the middle of the body and in the head, respectively. The co-localization of rRNA structures involved in subunit-subunit interaction and in the binding of eIF-3 to the protuberance indicates that structural alterations in this region of the rRNA could be involved in preventing premature association of the 40 S N particle with the large ribosomal subunit.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Melander

Holmberg

Nygård

1997

Journal of Biological Chemistry

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We have analyzed the structure of 18 S rRNA in native 40 S subunits using chemical modification followed by primer extension. The native subunits were modified using the single-stranded specific reagents dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The modification pattern of the 18 S rRNA was compared to that obtained from 1 binds to 40 S subunits and prevents formation of unprogrammed 80 S ribosomes by inhibiting association of the 40 S and 60 S ribosomal subunits in the absence of mRNA. Initiation factor eIF-2 selects the specific initiator tRNA (MettRNA f ) and brings it to the 40 S subunit. The resulting 43 S pre-initiation complex binds mRNA with the help of a series of initiation factors. The 60 S subunit now joins the mRNA containing 48 S initiation complex in a reaction that requires an additional initiation factor (eIF-5) and is associated with the hydrolysis of GTP.Several of the initiation factors are found to be associated with the so-called native 40 S ribosomal subunits (40 S N ) in vivo (2). Most of these factors are present on the 40 S N particles in small quantities, but eIF-3 is present in stoichiometric amounts (2). Initiation factor 3 is a huge multisubunit protein with a total mass of approximately 0.7 MDa (3). The factor displays RNA binding properties, and one of its subunits can be cross-linked to 18 S rRNA in the 40 S⅐eIF-3 complex (4). This suggests that rRNA may, at least in part, be responsible for binding the factor to the small ribosomal subunit. However, the location of the eIF-3 interaction site in 18 S rRNA is not known.The ribosomal RNA is considered to be involved in various ribosomal functions such as A-and P-site-related activities and peptide bond formation (for a review see Ref. 5). In prokaryotes the rRNA is directly involved in the binding of initiation factors and mRNA during protein synthesis initiation (6 -9). Less is known about the functional role of rRNA in the eukaryotic ribosome, but studies using chemical cross-linking and chemical and enzymatic footprinting have indicated that the rRNA is involved in mRNA binding, subunit interaction, and binding of elongation factors (10 -12).We have previously studied the structure of 18 S rRNA in derived 40 S subunits prepared by dissociation of isolated 80 S ribosomes (11, 13). In contrast to the native subunits, derived particles are free from additional non-ribosomal proteins. In this report, we have compared the structures of 18 S rRNA in native and derived 40 S subunits using chemical modification. The two types of 18 S rRNAs showed distinct but limited structural differences. The role of the non-ribosomal proteins in altering the structure of the 18 S rRNA in the 40 S N particles is discussed. Nygård and Nika (14). Briefly, isolated monosomes (15) were suspended in 0.5 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl 2 , and 10 mM 2-mercaptoethanol. The material was layered onto continuous 10 -40% (w/v) sucrose gradients containing 0.35 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl ...

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Section: Discussionmentioning

confidence: 99%

“…We have previously studied the structure of 18 S rRNA in derived 40 S subunits prepared by dissociation of isolated 80 S ribosomes (11,13). In contrast to the native subunits, derived particles are free from additional non-ribosomal proteins.…”

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confidence: 99%

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Melander

Holmberg

Nygård

1997

Journal of Biological Chemistry

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“…This central reaction, termed peptidyl transferase, requires an intact 28S rRNA in combination with four ribosomal proteins in the 60S ribosome. Work based upon the prokaryotic model indicates that the 28S rRNA equivalent is a critical component of the basic catalytic unit (9,10). The effect of NO on peptidyl transferase activity has not been previously examined.…”

Section: Figurementioning

confidence: 99%

Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages

Cai¹,

Guo²,

Schroeder³

et al. 2000

The Journal of Immunology

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NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mechanisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein synthesis.

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“…For example, changes in the chemical accessibility of rRNA nucleotides due to ribosomal subunit association have been measured using purified mammalian ribosomes (Holmberg et al 1994). The effects of HCV IRES binding to purified 40S ribosomal subunits have also been examined (Otto et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Shenvi¹,

Dong²,

Friedman³

et al. 2005

RNA

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Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg 2+ is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg 2+ concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg 2+ that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg 2+ concentrations. In low Mg 2+ concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.

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Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes

Cited by 22 publications

References 43 publications

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

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