<i>In vitro</i>
            assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit

Agalarov, S. Ch.; Zheleznyakova, E. N.; Selivanova, Olga M.; Zheleznaya, L. A.; Matvienko, N. I.; Vasiliev, V.D.; Spirin, Alexander S.

doi:10.1073/pnas.95.3.999

Cited by 51 publications

(31 citation statements)

References 22 publications

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“…Each of the 30S subunit large rRNA domains (body, head, and platform) can fold separately in vitro (Agalarov et al 1998(Agalarov et al , 1999 driven by binding of r-proteins (Sykes and Williamson 2009;Shajani et al 2011). Interdomain interactions occur late in the assembly process to properly orient the domains in the mature structure.…”

Section: Mechanisms Of Yjeq To Assist Maturation Of the 30s Subunitmentioning

confidence: 99%

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

Jomaa¹,

Stewart²,

Mears³

et al. 2011

RNA

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YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 39 end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.

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Section: Mechanisms Of Yjeq To Assist Maturation Of the 30s Subunitmentioning

confidence: 99%

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

Jomaa¹,

Stewart²,

Mears³

et al. 2011

RNA

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“…Tertiary binding proteins require prior binding of at least one secondary binding protein before they can associate with the 16S rRNA. Interestingly, the three major domains in 16S rRNA can assemble independently to form ribonucleoprotein particles (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1998). Kinetic analysis of some of these RNPs has revealed the dynamics of 30S assembly (Recht and Williamson 2001).…”

Section: Introductionmentioning

confidence: 99%

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ghosh

Joseph

2005

RNA

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Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

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“…Also, in both the 59 and 39 major domains Fe(II)-S8 mediated cleavages in 30S subunits were absent in binary complexes. Restoration of cleavage patterns required the assembly of entire domains, highlighting the significance of long-range interactions in assembly, even between independent events (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1998Agalarov et al , 1999.…”

Section: Introductionmentioning

confidence: 99%

“…All figures containing three-dimensional structures were prepared using the PyMOL and the Protein Data Bank file 2AW7 (Schuwirth et al 2005). domain exhibits similar patterns of dependencies (see Fig. 1A) and can be assembled independently in vitro (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1998Agalarov et al , 1999.…”

Section: Introductionmentioning

confidence: 99%

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

Calidas

Culver²

2010

RNA

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The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 59 terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

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In vitro assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit

Cited by 51 publications

References 22 publications

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

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