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Proteins and RNA sequences required for the transition of the t-Utp complex into the SSU processome.

ABSTRACT: Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling highly structured rRNAs with ribosomal proteins. Transcription and processing of pre-rRNAs are linked by the transcription-Utp sub-complex (t-Utps), a sub-complex of the small subunit (SSU) processome and prompted the investigations for the requirements of t-Utp formation and transition into the SSU processome. The rDNA promoter, the first 44 nucleotides of the 5?ETS, and active transcription by pol I were sufficient to recruit the t-Utps to the rDNA. Pol5, accessory factor, dissociated as t-Utps matured into the UtpA complex which permitted later recruitment of the UtpB, U3 snoRNP and the Mpp10 complex into the SSU processome. The t-Utp complex associated with short RNAs 121 and 138 nucleotides long transcribed from the 5?ETS. These transcripts were not present when pol II transcribed the rDNA or in nondividing cells. Depletion of a t-Utp, but not of other SSU processome components led to decreased levels of the short transcripts. However, ectopic expression of the short transcripts slowed the growth of yeast with impaired rDNA transcription. These results provide insight into how transcription of the rRNA primes the assemble of t-Utp complex with the pre-rRNA into the UtpA complex and the later association of SSU processome components.

SUBMITTER: Gallagher JEG

PROVIDER: S-EPMC6255707 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Proteins and RNA sequences required for the transition of the t-Utp complex into the SSU processome.

Gallagher Jennifer E G JEG

FEMS yeast research 20190101 1

Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling highly structured rRNAs with ribosomal proteins. Transcription and processing of pre-rRNAs are linked by the transcription-Utp sub-complex (t-Utps), a sub-complex of the small subunit (SSU) processome and prompted the investigations for the requirements of t-Utp formation and transition into the SSU processome. The rDNA promoter, the first 44 nucleotides of the 5΄ETS, and active transcription by pol I ...[more]

PMID: 30445532

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Project description:3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Dataset Information

Proteins and RNA sequences required for the transition of the t-Utp complex into the SSU processome.

Publications

Proteins and RNA sequences required for the transition of the t-Utp complex into the SSU processome.

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