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N D Denslow

Publications and source records attributed to N D Denslow.

45 records · Page 3Linked to original sources
PubMed1989

Mechanism of mRNA binding to bovine mitochondrial ribosomes.

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.

Animals↗
PubMed1988

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3).

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.

Animals↗
PubMed1988

RNA binding proteins of the large subunit of bovine mitochondrial ribosomes.

RNA binding properties of proteins from the large subunit of bovine mitochondrial ribosomes were studied using four different approaches: binding of radiolabeled RNA to western blotted proteins; disassembly of the intact 39 S ribosomal subunits with urea; binding of ribosomal proteins to RNA in the presence of urea; and binding of proteins extracted with lithium chloride to RNA. Results from these four approaches allowed us to identify a set of six proteins (L7, L13, L14, L21, L26, and L44) which appear to be strong RNA binding proteins. Seven additional proteins (L8, L11, L28, L35, L40, L49, and L50) were identified as secondary RNA binding proteins. RNA binding properties of the proteins in both of these sets were compared with the topographic disposition and susceptibility towards lithium chloride extraction of the individual proteins. Proteins from the first set are good candidates for early assembly proteins since they have a high affinity for RNA, are generally found in 4M lithium chloride core particles, and are among the most buried proteins in the 39 S subunit.

Animals↗
PubMed1984

Organization of proteins in mammalian mitochondrial ribosomes. Accessibility to lactoperoxidase-catalyzed radioiodination.

To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, we used a double label iodination technique. Regions of r-proteins exposed in purified ribosomal subunits were labeled with 131I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with 125I using the chloramine-T mediated reaction. The ratio of 131I to 125I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the "highly exposed" group, and a large number of proteins in the "intermediate exposure" group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as "assembly proteins," since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles.

Animals↗
PubMed1982

Protein composition of the bovine mitochondrial ribosome.

The protein complement of the bovine mitochondrial ribosome has been analyzed by two-dimensional electrophoresis in polyacrylamide gels to determine the number and molecular weights of the ribosomal proteins. Salt-washed ribosomal subunits are found to contain a total of 85 ribosomal proteins, 84 of which are electrophoretically distinct between the two subunits. These proteins are also electrophoretically distinguished from those of cytoplasmic ribosomes. This large number of proteins does not appear to be due to contamination by cytoplasmic ribosomal proteins or by adherent nonribosomal proteins. The molecular weights of these proteins are considerably larger than those of Escherichia coli ribosomal proteins, and are similar to those of bovine cytoplasmic ribosomal proteins. The sum of the molecular weights of the 85 proteins agrees well with that predicted by physical chemical measurements of the total mass of protein in the two subunits. Bovine mitochondrial ribosomes thus contain about twice as much protein as RNA, a highly unusual composition in comparison to the other kinds of ribosomes which have been characterized to date. In addition, it appears that the ribosomal proteins themselves are less basic than the proteins of most other ribosomes.

Animals↗
PubMed1978

Antibiotic susceptibility of the peptidyl transferase locus of bovine mitochondrial ribosomes.

We have a used a modified 'fragment reaction' to compare the susceptibility of isolated bovine mitochondrial ribosomes, bacterial (Escherichia coli) ribosomes and eukaryotic (bovine microsomal) ribosomes to several antibiotics. All of these ribosomes share certain structural features of their peptidyl transferase center, as indicated by their interaction with substrates, puromycin and gougerotin. Bovine mitochondrial ribosomes have other structural features in common with bacterial, but not cytoplasmic ribosomes, as revealed by their susceptibility to chloramphenicol and the streptogramin antibiotics. While mitochondrial ribosomes are susceptible to all inhibitors of bacterial ribosomes tested, their low susceptibility to the lincosamines and macrolides suggests that some component(s) of the binding sites for these antibiotics is altered.

Acyltransferases↗