tRNA-assisted overproduction of eukaryotic ribosomal proteins.

Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.

A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB.

Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B". We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature-sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC-DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA complex. The two-hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B" component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B". An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.

A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

The largest subunits of the three eukaryotic nuclear RNA polymerase present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co-linear regions. Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases. An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single-site substitutions. A few conditional mutations were also obtained. One of them, rpc160-112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C. RNA polymerase III from the mutant rpc160-112 was severely impaired in its ability to transcribe a tRNA gene in vitro. The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate. Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild-type enzyme. The enzyme defect was also detectable with poly[d(A-T)] as template, in the presence of saturating DNA, ATP and UTP concentrations. The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product.

Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide growth-arrested yeast cells.

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

Cardiac pacing in unilateral left superior vena cava: evaluation by digital angiography.

In a patient with complete heart block and chronic lymphocytic leukemia a pacemaker lead could not be introduced from either the right or left subclavian vein. Digital subtraction angiography excluded a neoplastic mediastinal mass, demonstrated a unilateral left superior vena cava and defined the best route for lead insertion.

[Continent cecal-colonic reservoir. Surgical technique]. / Serbatoio cieco-colico continente. Technica operatoria.

The Authors discuss a recent case report treated with radial cystectomy associated with a secondary urinary derivation using the caecum-colon reservoir. After having reviewed the various surgical procedures involving the urinary derivations, the Authors describe the technique used by them paying particular attention to the positive aspects of having a low filling pressure reservoir controlled by a valid sphincter ileum-caecum valve. Considering the good postoperative result with this method, the Authors regard this procedure as an alternative to other urinary derivation techniques when carried out with correct indications.