Loss of a nonenzymatic function of XPG results in defective transcription-coupled repair (TCR), Cockayne syndrome (CS), and early death, but the molecular basis for these phenotypes is unknown. Mutation of CSB, CSA, or the TFIIH helicases XPB and XPD can also cause defective TCR and CS. We show that XPG interacts with elongating RNA polymerase II (RNAPII) in the cell and binds stalled RNAPII ternary complexes in vitro both independently and cooperatively with CSB. XPG binds transcription-sized DNA bubbles through two domains not required for incision and functionally interacts with CSB on these bubbles to stimulate its ATPase activity. Bound RNAPII blocks bubble incision by XPG, but an ATP hydrolysis-dependent process involving TFIIH creates access to the junction, allowing incision. Together, these results implicate coordinated recognition of stalled transcription by XPG and CSB in TCR initiation and suggest that TFIIH-dependent remodeling of stalled RNAPII without release may be sufficient to allow repair.

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.

The multisubunit transcription factor TFIID is essential for directing eukaryotic promoter recognition and mediating interactions with activators/cofactors during assembly of the preinitiation complex. Despite its central role in transcription initiation and regulation, structural knowledge of the TFIID complex has so far been largely limited to electron microscopy studies of negatively stained samples. Here, we present a cryo-electron microscopy 3D reconstruction of the large endogenous human TFIID complex. The improved cryopreservation has allowed for a more detailed definition of the structural elements in the complex and for the detection, by an extensive statistical analysis of the data, of a conformational opening and closing of the cavity central to the TFIID architecture. We propose that these density rearrangements in the structure are a likely reflection of the plasticity of the interactions between TFIID and its many partner proteins.

U12-dependent introns containing alterations of the 3 splice site AC dinucleotide or alterations in the spacing between the branch site and the 3 splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5 splice site AU dinucleotide, any nucleotide could serve as the 3-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3 splice site function. A branch site-to-3 splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3 splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3 splice site but that formation of the prespliceosomal complex A requires an active 3 splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3 splice site.Two types of spliceosomal introns are known to exist in higher eukaryotic plants and animals (see reference 6 for a recent review). The major class, termed here the U2-dependent class, has been the subject of a great deal of investigation for many years. The minor or U12-dependent class has been recognized only relatively recently. Many genes contain both types of introns in an interspersed pattern, requiring cooperation between the two splicing systems to properly identify the exons. The size distributions of introns and the adjacent exons are similar for both classes of introns. This suggests that the process of splice site recognition for both classes must contend with similar problems of distinguishing correct splice sites amidst the often many tens or hundreds of thousands of nucleotides in a pre-mRNA.The information needed to specify the sites of splicing of both classes of introns is largely located at or near the 5Ј and 3Ј splice sites. These regions contain conserved sequences that interact with the splicing machinery to promote the assembly of the spliceosome and to specify and activate the chemical cleavage and ligation reactions which lead to the production of spliced RNA. The 3Ј splice site region contains two nucleotides that must be precisely located and activated for the two chemical steps in the splicing reaction: the branch site adenosine, with its associated 2Ј hydroxyl group, which is the nucleophile in the first step of splicing, and the 3Ј splice site residue, which lies immediately adjacent to the phosphodiester bond that is transesterified in the second step of splicing. The 3Ј splice site is physically separate from the branch site residue and is not involved in the chemistry of the first-step reaction. Indeed, several studies have shown that the first splicing step can occur in vitro on RNA molecules in which no active 3Ј splice site is present due to either truncation of the substrate RNA (1, 13, 33) or mutation of the 3Ј splice site AG (15,31,39).In U2-dependent introns, the branch site aden...

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The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The ‘G’ class of RNP complexes consists of 170‐300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing‐like activity. The ‘T’ class consists of approximately six RNP complexes, the endogenous RNA of which can be self‐labeled with [alpha‐32P]UTP. The most abundant T complex, T‐IV, is visualized by electron microscopy as 80‐140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.

We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from ϳ160 to ϳ60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.RNA-binding proteins are involved in the synthesis, processing, transport, translation, degradation, and stabilization of RNA (7, 24). The mitochondrial genome of trypanosomatids is composed of thousands of circular DNA molecules, which are catenated and form a dense body of DNA, called the kinetoplast DNA network. This consists of two types of molecules: maxicircles and minicircles. The maxicircle encodes two rRNAs, 18 mRNAs, and a few gRNAs (2). The minicircle encodes the majority of the gRNAs (28). The maxicircle protein coding regions are very compact, and many of the genes overlap extensively, leaving little room for regulatory sequences that might control transcription and RNA processing. It was shown previously that maxicircle transcription produces polycistronic RNAs that are processed to mature RNAs (16,27). The presence of such precursors indicates that the mature RNAs are generated by 3Ј-and 5Ј-end processing, which, for several of the overlapping genes, eliminates a portion of the coding regions of the adjacent transcripts, which then must be degraded. Mitochondrial biogenesis in Trypanosoma brucei is developmentally regulated (23,26,29). In long slender bloodstream forms mitochondrial function is strongly repressed.During differentiation, the mitochondrion is partially activated in the short stumpy bloodstream form, but only in the insect midgut procyclic form is the mitochondrion fully active. This change in physiology is reflected in a stage-specific regulation of mitochondrial gene expression (15, 18). The levels of some mitochondrial transcripts in short stumpy cells are intermediate between the long slender and procyclic forms, whereas other transcripts are already at the procyclic levels. The transcription rates and processing of the rRNA genes are the same in the bloodstream and the procyclic forms, suggesting that the difference in abundance of the mature rRNAs is due to differential stability in the two forms of the life cycle (19). T...