Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.

The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 M.In eukaryotes, ribosome biogenesis requires the coordinated processing and assembly of four ribosomal RNAs (rRNAs) and about 78 ribosomal proteins (49). In Saccharomyces cerevisiae, the 35S pre-rRNA is transcribed by RNA polymerase I in the nucleolus and is cleaved in several places to produce the mature 18S, 5.8S, and 25S rRNAs (Fig. 1). This process utilizes over 180 trans-acting factors and yet occurs fast enough to allow 2,000 ribosomes to be made each minute (for reviews, see references 16, 17, 22, 24, and 51). In this highly coordinated process, many factors assemble onto the nascent pre-rRNA as it is transcribed, forming large ribonucleoproteins (RNPs) that mature the small subunit (SSU) or large subunit (LSU) of the ribosome. Despite the identification of such trans-acting factors, the details of ribosome biogenesis remain elusive, as the complexity of ribosome biogenesis lies in the folding of prerRNAs and ribosomal proteins into functional ribosomes. Indeed, the frontier of ribosome synthesis investigation is to delineate the role(s) of each trans-acting factor in the production of the rRNA as it is modified, processed, and folded to become the mature ribosome (22).Ribosome biogenesis is a dynamic process in which transacting factors associate with and dissociate from the evolving pre-rRNA throughout its maturation. The initial assembly of factors involved in both SSU and LSU biogenesis with the 35S pre-rRNA has been termed the 90S preribosome, which is separated by cleavage in ITS1 into the SSU processome (required for SSU maturation) and the 66S preribosome (required for LSU maturation) (22). An RNP forming around the pre-18S rRNA has long been visualized in Miller chromatin spreads as a packed structure (35)

Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.

The half-a-tetratricopeptide (HAT) repeat motif is of interest because it is found exclusively in proteins that are involved in RNA metabolism. Little is known about structure-function relationships in this class of repeat motif. Here, we present the results of a combined bioinformatics, modeling and mutagenesis study of the HAT domain of Utp6. We have derived a new HAT consensus, delineated its structure-defining residues and, by homology modeling, have placed these residues in a structural context. By considering only HAT motifs from Utp6 proteins, we identified residues that are shared by, and unique to, only this subset of HAT motifs, suggesting a key functional role. Employing both random and directed mutagenesis of the HAT domain in yeast Utp6, we have identified residues whose mutation results in loss of function. By examining these residues in the context of the homology model, we have delineated those that act by perturbing structure and those that more likely have a direct effect on function. Importantly, the residues we predict to have a direct effect on function map together on the tertiary structure, thus defining a potential functional interaction surface.

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