The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.

Well-ordered three-dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)-system.

A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.The secondary structure of the 16S RNA of the small 30S ribosomal subunit is divided into three major domains (1). Each domain and a defined set of ribosomal proteins are involved in the formation of different morphological parts of the subunit. The 5Ј domain corresponds to the central body of the 30S subunit, the central domain is involved in the formation of the side bulge or platform, as well as part of the body, and the 3Ј major domain is localized in the head region of the subunit (2-4).It has been shown that fragments of the 16S RNA corresponding to the domains of the 16S RNA are capable of assembling in vitro with specific sets of ribosomal proteins into compact ribonucleoprotein particles (RNPs). Ofengand and coworkers (5) demonstrated formation of the RNP containing the 5Ј domain of the 16S RNA and four ribosomal proteins. Noller and coworkers (6) showed that the fragment of the 16S RNA, corresponding to the 3Ј domain of the 16S RNA, assembles with the group of eight ribosomal proteins. It is noteworthy that the proteins bound in the both complexes are the same as those found associated with these domains in the intact 30S subunit. In this work, we describe the assembly of an RNP containing the central domain of the 16S RNA and five ribosomal proteins of the Thermus thermophilus ribosome.The sequence of the 16S RNA from T. thermophilus was determined, and a high homology of the sequences of the 16S RNA from T. thermophilus and Escherichia coli was shown (7). The homology is 75%, and the computer model of its secondary structure (Fig. 1) does not differ from the model of the E. coli 16S RNA (8). The ribosomal proteins of the 30S subunit of T. thermophilus were purified and identified both by twodimensional gel electrophoresis and amino-terminal sequence analysis (9, 10). The homology of 17 of 19 30S ribosomal proteins from T. thermophilus with corresponding proteins of E. coli was established. Thus the results presented herein are interpretable within the framework of the well-known findings conce...

Fragments of the 16S rRNA of Thermus thermophilus representing the 3 H domain (nucleotides 890±1515) and the 5 H domain (nucleotides 1±539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3 H RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3 H domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5 H RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit Keywords: 30S ribosomal subunit; domain structure of ribosomal particles; reconstitution of ribosomal domains; ribosomal proteins; rRNA.The 16S RNA of the small ribosomal subunit is divided into three major domains [1]. Each domain and a defined set of ribosomal proteins are involved in the formation of different morphological parts (lobes) of the subunit. The 5 H domain (nucleotides 1±563) corresponds to the central body of the 30S subunit, the central domain (nucleotides 564±915) is involved in the formation of the side bulge or platform, whereas the 3 H major domain (nucleotides 919±1396) is localized in the head region of the subunit; there is also an additional 3 H minor domain (residues 1397±1542).It has previously been shown that the isolated fragments of the 16S RNA corresponding to its major domains are capable of assembling in vitro with specific sets of ribosomal proteins into compact RNPs. Ofengand and co-workers [2] demonstrated the formation of RNP containing the 5 H domain of Escherichia coli 16S RNA and four ribosomal proteins. Noller and co-workers [3] showed that the fragment of E. coli 16S RNA comprising the 3 H domain assembles with a group of eight ribosomal proteins. The proteins bound in both complexes were the same as those found to be associated with these domains in the intact 30S subunit. Recently we have shown formation of RNP containing the central domain of the 16S RNA from Thermus thermophilus and five ribosomal proteins [4].In this paper we report on the assembly of RNP particles consisting of the 5 H and 3 H domains of the T. thermophilus 16S rRNA and corresponding sets of ribosomal proteins from this bacterium.

Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way

The recombinant mRNAs with 5′-untranslated region, called omega leader, of tobacco mosaic virus RNA are known to be well translated in eukaryotic cell-free systems, even if deprived of cap structure. Using the method of primer extension inhibition (toe-printing), the ribosomal particles were shown to initiate translation at uncapped omega leader when its 5′-end was blocked by a stable RNA-DNA double helix, thus providing evidence for internal initiation. The scanning of the leader sequence and the formation of ribosomal 48S initiation complexes at the initiation AUG codon occurred in the absence of ATP-dependent initiation factor eIF4F, as well as without ATP. The latter results implied the ability of ribosomal initiation complexes for ATP-independent, diffusional wandering (also called bi-directional movement) along the leader sequence during scanning.

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