METHODS 25, 289–291 (2001)doi:10.1006/meth.2001.1240, available online at http://www.idealibrary.com on

EDITORIAL

Structure Determination of Ribosomes andOther Complex Ribonucleoproteins

Ribosomes, the ribonucleoprotein machines responsible for protein synthesisin all cells, have been the subject of intense study for more than 40 years. Avariety of biochemical and structural techniques have been brought to bearon the problem of understanding the molecular architecture of the ribosome,starting with the sequencing of the ribosomal RNAs and the identification ofthe protein components in the 1960s and the 1970s, to detailed electron densitymaps derived using cryo-electron microscopy (cryo-EM) in the late 1990s.

This steady progress has given way to a dramatic leap forward in our under-standing of the ribosomal structure over the past few years, as the X-ray crystal-lographic efforts, first started in 1980 by Yonath and Wittmann, have finallyborne fruit. The first low-resolution atomic structures of the 50S subunitemerged in 1998, aided by lower-resolution cryo-EM maps. This was followedin rapid succession by more detailed atomic structures of the two individualsubunits, as well as the whole 70S prokaryotic ribosome in 1999–2000 (reviewedby Ramakrishnan and Moore (2001), Curr. Opin. Struct. Biol. 11, 144–154).

These events have dramatically changed and energized the whole field ofribosomal study. As observed by Peter Moore at the 1999 ribosome meeting,1

these detailed structures mark the “end of the beginning” and the start of anew quantitative age in the study of ribosomal structure and function. Theatomic resolution structures emerging from X-ray crystallography are now pro-viding a framework that will allow a more detailed examination of the intricatechoreography of ribosomal components and their conformational changes thatis at the heart of how all cells make proteins from mRNAs.

This issue of Methods aims to describe some of the techniques and approachesthat will be important as we move into this new era of ribosome structural andfunctional studies. Both physical and biochemical approaches to the study ofribonucleoprotein complexes are discussed.

Physical ApproachesThe first two articles relate to issues involving X-ray crystallography of large

macromolecular complexes such as the ribosome. Enormous hurdles had to beovercome to obtain the first atomic resolution structures for the ribosomal

1 Helsingør Conference, Helsingør, Denmark, June 13–17, 1999. in The Ribosome: Structure,Function, Antibiotics, and Cellular Interactions (Garrett, R. A., Douthwaite, S. R., Liljas, A.,Matheson, A. T., Moore, P. B., and Noller, H. F., Eds.), ASM Press, Washington, DC, 2000.

1046-2023/01 $35.00 289q 2001 Elsevier ScienceAll rights reserved.

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subunits. Many of these hurdles and approaches to improving crystals andusing large heavy metal clusters for phasing are described by Yonath andcolleagues. The article by Cate describes how initial low-resolution phases forcrystallographic data were obtained using results from cryo-electron micros-copy, and the subsequent use of multiwavelength anomalous dispersion ap-proaches to extend these phases to higher resolution.

Electron microscopy has played an indispensable role in our understandingof ribosomal structure, from the days of rough shapes of the individual subunitsto the current detailed electron density maps where even some atomic featurescan be discerned. Although X-ray crystallography is better suited for detailedatomic resolution mapping of macromolecular complexes, cryo-electron micros-copy is playing a very important role in mapping the different dynamic statesand subunit/tRNA/factor interactions that are required for each amino acidaddition during the process of translation. The article by Frank discusses someof the methodological issues involved in the use of cryo-EM for understandingthe dynamic states of the ribosome.

Unlike X-ray crystallography, nuclear magnetic resonance (NMR) is bestsuited for the study of smaller molecular systems. Nevertheless, NMR canprovide valuable data on the dynamics and motion of molecular components.Properly designed RNA analogs can often yield significant functional and struc-tural insights into important regions of large ribonucleoprotein complexes suchas the ribosome. The article by Lukavsky and Puglisi details some techniquesfor rapid NMR structure determination of RNA oligonucleotides.

Biochemical ApproachesThe next two articles describe two powerful techniques for probing structural

relationships among RNA molecules in and on the ribosome. Crosslinking hashad a long and honorable history of providing information on close contactsbetween two components when properly controlled, and Wollenzien and col-leagues illustrate this with two procedures for the study of such close contactsboth within the ribosome and between RNA of the ribosome and liganded tRNAor mRNA. Both random (by direct UV irradiation) and directed (by specificplacement of 4-thiouridine) methods are described. Bowen et al. compare thetwo chemical nucleases 1,10-phenanthroline–Cu(II) and EDTA–Fe(II). Al-though both act by generating chemical radical species that cleave RNA chains,they vary in terms of their specificity and range of action. When they areattached to oligodeoxynucleotides that are then hybridized to RNA, their cleav-age action can be exploited for analysis of neighboring RNA segments in acomplex tertiary structure. Although both articles focus their application on theribosome, the techniques are equally applicable to any large ribonucleoproteincomplex such as the spliceosome.

RNA–protein interaction is the subject of the article by Stelzl and Nierhaus.They describe a very effective SELEX variant called SERF which is used toidentify those segments of a large RNA that specifically interact with a particu-lar protein of interest. This is just the situation that occurs in the ribosome,where ribosomal proteins bind to particular and often noncontiguous regionsof a single large RNA molecule. It is an alternative to the various types ofprotection assays that have been used previously, and can be used for the studyof any large ribonucleoprotein complex.

Moine et al. describe an in vivo selection procedure for detecting all possiblenucleotide substitutions in any chosen small segment or segments of a ribo-somal RNA that will still allow ribosomal function as measured by cell growth.The value of this approach, termed by the authors “artificial phylogeny,” isthat it allows exploration of all possible nucleotides at a given site or all possible

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combinations at two sites that are functional, whereas the variations introducedamong species by natural selection may not be all-inclusive for a variety ofreasons.

Ribosomal RNA, tRNA, and even spliceosomal RNA and small nucleolarRNAs contain modified nucleotides, mainly pseudouridine and 28-O-methylatednucleosides, which, while ubiquitous, have no known function. Knowledge oftheir location in RNAs is an essential prerequisite to understanding their role.Classic methods of RNA sequence analysis are extremely tedious, particularlyso for pseudouridine, and especially in large RNAs like the ribosomal RNAs.The last two articles describe rapid methods for sequencing these two classesof modified nucleosides. Ofengand et al. detail a reverse transcriptase procedurefor mapping pseudouridines in ribosomal RNA and tRNA, and Maden surveysthe current rapid methods for locating 28-O-methyl nucleosides.

An issue of this size cannot possibly cover all the important techniquesand methods for the study of ribonucleoprotein complexes. Other structuraltechniques including low-angle X-ray scattering, conventional cryo-EM tech-niques, RNA model building and refinement approaches, as well as single-molecule and scanning electron microscopy (SEM) approaches, have not beendiscussed in this issue. Additional biochemical approaches include chemicalprobing of RNA structure, matrix-assisted laser desorption/ionization (MALDI)mass spectroscopic analysis of RNA–protein crosslinks, fluorescence resonanceenergy transfer, and nucleotide analog interference mapping. Another areathat will become increasingly important as details of the translational machin-ery are uncovered in atomic detail is careful kinetic and mechanistic studiesof the translational cycle. Some of these and other techniques relevant to thestudy of ribonucleoprotein complexes have been described in Volumes 317 and318 of Methods in Enzymology, as well as in Methods, Volumes 18(1) and 23(3).

Although the articles in this issue describe methods being used for the studyof ribosomes, these approaches are also suitable for studying other ribonu-cleoprotein complexes such as those involved in splicing and protein export/transport.

Arun MalhotraJames OfengandGuest Editors