Localization of riboproteins in a trypanosomatid mitochondrion.

There is growing evidence in support of mitochondrial translation in trypanosomes but mitoribosomes have never been characterized or localized in these parasites. On RNA-protein blots we identified several proteins from the trypanosomatid Crithidia fasciculata which bound the parasite's 12S and 9S mitochondrial ribosomal RNAs. Two of these proteins had significant amino acid sequence homology to riboproteins S8 and S21 across phyla. Immunoelectron microscopy revealed that antibodies raised against the two proteins react with matrix components in the C. fasciculata mitochondrion. Our data thus provide, we believe for the first time, evidence for the presence of riboproteins within a trypanosomatid mitochondrion, bound, possibly, to the 12S and 9S RNAs. The proteins were immunologically related to two cytosolic riboproteins which were also of identical size, suggesting the interesting possibility that the same set of riboproteins is shared between the cytosol and the mitochondrion in this parasite.

Protein synthesis in mitochondria isolated from the trypanosomatid protozoan Crithidia fasciculata.

Evidence presented over the years in support of mitochondrial translation in trypanosomes, based largely on studies using differential inhibitors such as cycloheximide and chloramphenicol, remains controversial. I have studied endogenous mRNA-dependent translation in a mitochondrial fraction isolated from the trypanosomatid protozoan Crithidia fasciculata. By using pancreatic ribonuclease to inactivate contaminating cytosolic activity, I show that these mitochondria can conduct protein synthesis in their own right. The mitochondrial translational products differed from cytosolic products as judged by SDS-PAGE, and had sizes expected of some proteins encoded in the mitochondrial genome of C. fasciculata and other trypanosomatids. Some evidence is provided suggesting that the seat of translation might be the kinetoplast.

Evidence for close in vivo association of protein with mitochondrial ribosomal RNA in a trypanosome.

Riboprotein particles containing the ribosomal RNA-like 9S and 12S RNAs in the trypanosome mitochondrion have never been isolated. Using the formaldehyde cross-linking procedure I show here that one or more of three proteins, q2 (16.5 kDa), r (15 kDa) and s (13 kDa), are closely associated in vivo with the 9S and 12S RNAs of the trypanosomatid parasite, Crithidia fasciculata. These proteins were also found to be associated with the parasite's cytosolic ribosomal RNA and to have strong immunological cross reactivity with riboprotein S11 of the bacterium Escherichia coli. These data provide the first evidence for the association of riboprotein-like proteins with the 9S and 12S mitochondrial RNA in a trypanosome, possibly as components of a mitochondrial ribosome.

Two proteins involved in kinetoplast compaction.

The kinetoplast is the genome of the single mitochondrion of trypanosomatid Protozoa, and contains up to 30% of total cellular DNA in a network of catenated AT-rich rings. EM studies show that the kinetoplast is organized into a compact, disc-shaped structure in vivo, but little is known about proteins involved in its architecture. Defining such proteins would be useful to understand the molecular biology of this unusual organelle and to design compounds to contain parasite growth. We show here that two proteins, p1 and p2 of M(r) approximately 22 and approximately 21 kDa, respectively, from the trypanosomatid Crithidia fasciculata can compact kDNA networks efficiently in vitro, the first such demonstration with purified trypanosome proteins. We show that these proteins are localized exclusively in the parasite's kinetoplast. Our data thus define two proteins potentially involved in kinetoplast organization in vivo.

Identification of DNA-binding proteins in the parasitic protozoan Crithidia fasciculata and evidence for their association with the mitochondrial genome.

The single mitochondrion of trypanosomatid protozoans such as Crithidia fasciculata has a large and complex network of AT-rich DNA called kDNA. Little is known about proteins involved in the packaging and morphogenesis of this network. I report the purification of a protein fraction from this parasite which preferentially retained kDNA in the gel slot during electrophoresis. The proteins had approximate molecular weights of 22, 21, 17.5, 16.5, 15, and 13 kDa. They copurified with mitochondrial DNA, parts of which they protected from nuclease attack. The proteins resembled histones but they also differed from histones in significant ways. Immunological evidence showed that the proteins were enriched in a mitochondrial fraction, implying their association with kDNA in vivo.

A simple procedure for detecting proteins that bind preferentially to kDNA networks.

A procedure is described in which a fractionated protein extract from the trypanosome Crithidia fasciculata was incubated either with deproteinized C. fasciculata kDNA networks or nuclear DNA. At least three proteins in the range of 45-65 kDa had a specific affinity for the networks. We suggest that these proteins may play an important role in the biology of the kinetoplast.

Variable RNA polymerase populations in the life cycle of Trypanosoma brucei.

DNA-dependent RNA polymerase from blood forms and culture forms of the parasitic protozoan Trypanosoma brucei was resolved into multiple peaks of activity by DEAE-Sephadex chromatography. The enzyme from the two forms was found to be different by several criteria, suggesting that it is subject to developmental control.

Salmonella typhimurium rpoB and rpoC genes cloned on lambda phages.

Salmonella typhimurium rpoB and rpoC genes, coding for the RNA polymerase subunits beta and beta', respectively, were isolated on lambda phages. Like their E. coli counterparts the two genes are closely linked and probably share a common promoter. The distribution of the target sites for several restriction enzymes, however, shows considerable divergence from the E. coli pattern.

Mutant of Escherichia coli with unusual patterns of rpoB,C expression in response to rifampicin and acridine orange.

A mutation located near rpoB (89') in E. coli is responsible for unusual patterns of beta and beta ' (but not L7/L12) synthesis in response to the drugs rifampicin and acridine orange.

Evidence for clustering of RNA polymerase and ribosomal protein genes in six species of Enterobacteria.

32P-labelled DNA fragments from the E. coli beta operon were used as hybridisation probes for homologous DNA sequences in chromosomal digests of six species of Enterobacteria. In all species the hybridisation pattern suggested clustering of the genes comprising the beta operon.

Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro.

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.

Escherichia coli merodiploids with altered ratios of episomal to chromosomal RNA polymerase activity.

Starting with an E. coli merodiploid strain (rpoB+ (rifS)/rpoB- (rifR)) containing equal amounts of rifampicin-sensitive and resistant RNA polymerase activities, mutants were isolated that had a disproportionately high ratio of the rifampicin-resistant enzyme activity. With one strain it is shown that the mutation responsible for this phenotype is closely linked to the rifR (rpoB-) allele.

Evidence against autorepression of the betabeta' operon in Escherichia coli.

By using rifampicin to increase the rate of beta and beta' synthesis in a heterodiploid strain of E. coli carrying the mutation rifpr (Km7), which codes for a rifampicin sensitive RNA polymerase to which the drug binds weakly, and the dominant mutation rifRD, which codes for a rifampicin resistant RNA polymerase, the concentration of these subunits in the cell was increased 1.6 fold. Measurements made after removal of rifampicin from the cells showed that the excess beta and beta' subunits did not reduce the rate of their own synthesis below normal.

Evidence for specific control of RNA polymerase synthesis in Escherichia coli.

Studies on the synthesis of RNA polymerase in E. coli rif mutants containing both sensitive and resistant RNA polymerase molecules show that the synthesis of E. coli RNA polymerase is under a specific and active control system.