Max Herzberg

Mouse nuclear satellite dna, 5-methylcytosine content, pyrimidine isoplith distribution and electron microscopic appearance.

Abstract Nuclear satellite DNA of high specific activity labelled with [ 3 H- methyl ]- l -methionine and 32 P was isolated from newborn mice and from cultured mouse embryo cells. This DNA was found to have more than twice the molar concentration of 5-methylcytosine than the main band of nuclear DNA. Labelled pyrimidine isopliths were separated by column chromatography on DEAE cellulose. The distribution of radioactivity in such isopliths did not reveal any feature to distinguish nuclear satellite DNA from the main band DNA. Visualization of renatured satellite DNA by electron microscopy revealed a population of linear molecules of up to 4.0 μ in length, distributed into four groups with average lengths of 0.9, 1.8, 2.7 and 3.6 μ. Loops of approximately 0.2 μ in circumference were seen at intervals along the molecules.

Purification of initiator C from Escherichia coli: a protein which binds messenger RNA and initiator tRNA to the 30 s ribosome.

Attachment of 30 s ribosomes to nascent messenger RNA, which can be studied by electron microscopic quantitation of the number of ribosomes bound to bacteriophage T4 DNA transcribed with RNA polymerase, requires the presence of initiator protein C(F2). On the other hand, C also promotes the binding of N-formylmethionyl-tRNA to the 30 s ribosome. Evidence is presented that both mRNA and initiator tRNA binding activities are due to the same protein.

Size determination of mitochondrial ribosomal RNA from Aspergillus nidulans by electron microscopy

Abstract Mitochondrial ribosomal RNA from Aspergillus nidulans has been visualized in the electron miscroscope and compared to its homologous cytoplasmic ribosomal RNA and to Escherichia coli ribosomal RNA. The mean chain lengths of the two mitochondrial ribosomal RNA components are found to be 0.47 and 0.91 μ, whereas those of cytoplasmic ribosomal RNA are 0.52 and 1.10 μ and those of E. coli ribosomal RNA are 0.40 and 0.72 μ. Based upon its length, mitochondrial ribosomal RNA appears larger in size than bacterial-type ribosomal RNA but smaller than cytoplasmic ribosomal RNA.

[28] Escherichia coli initiation factors in the binding of ribosomes to messenger RNA

Publisher Summary Purified ribosomes are incapable of initiating the translation of messenger RNA (mRNA) unless initiation factors are added, and in the absence of these proteins, the mRNA chain does not even become attached to the ribosomes. This chapter describes the preparation of ribosomes and initiation factors for two different methods that are useful to investigate the role of initiation factors in the binding of ribosomes to mRNA. One method uses a combination of zone sedimentation and membrane filtration to detect the binding of labeled mRNA to ribosomes. While, the other is a more rapid and quantitative assay, which measures by direct electron microscopic observation, the attachment of ribosomes to DNA-bound nascent mRNA. The sedimentation of radioactive mRNA is studied on linear density gradients in which messenger RNA binds to ribosomes sediments more rapidly than free mRNA. The direct electron microscopic observation method is rapid and allows simultaneous quantitative comparison of many samples.

Translation initiation factor C (f2): Selective inactivation of its f-met-tRNA binding activity which does not affect messenger RNA binding to the 30 S ribosome.

Initiation factor C (f2) stimulates both the binding of messenger RNA to the 30 S ribosome [ 1,3] and the attachment of formyl-methionyl tRNA (f-mettRNA) to this subunit [2,3]. Attempts to separate these two activities during the purification procedure [3] have remained unsuccessful. The mRNA 30 S interaction promoted by factor C (f2) does not, however, require the presence of f-met-tRNA [4] . The two activities of the factor seem therefore to be independent from each other, suggesting that the protein might have multiple active sites. Recently, Mazumder et al. [5] reported that factor f2 (C) possesses an essential sulfhydril group whose blocking produced a complete inactivation of the effect of the factor on f-met-tRNA binding to the ribosome. The present work shows that factor C dependent f-met-tRNA binding is indeed abolished after treatment of the protein with N-ethyl maleimide (NEM) but the activity of the factor for messenger RNA binding to the 30 S ribosome is not impaired. These findings support the notion that different sites in the protein are responsible for f-mettRNA and mRNA binding.