Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
The Eukaryotic Chromosome and Its Control
Nuclear Proteins

In bacterial Cells, the negatively charged phosphate groups of DNA can be largely neutralized by positively charged Polyamines. However, basic Proteins also tend to partially "coat" DNA. In the mature heads of fish spermatozoa, densely packed DNA is neutralized by protamines—specialized low-molecular-weight proteins (with a Molecular Weight of ~5000) rich in Arginine residues. Similar basic proteins have been found in mammalian sperm [284]. In somatic cells, however, the negative charges of DNA are compensated primarily by the positive charges of a heterogeneous group of basic proteins known as Histones. There are five classes of histones, with molecular weights ranging from ~11,000 to 21,500 [285–287]:

H1 (or I, or f1)

Lysine-rich

H2a (or IIb1, or f2a2)

H2b (or IIb2, or f2b)

Moderately lysine-rich

H3 (or III, or f3)

H4 (or IV, or f2a1)

Arginine-rich

A hallmark of arginine-rich histones is the remarkable constancy of their Amino Acid Sequence. For instance, histone H4 from pea seedlings differs from the analogous histone in bovine Thymus by only Two Amino Acids. As for the lysine-rich histone H1, its sequence exhibits a high degree of species Specificity.

Calf thymus histone H3 contains 135 amino acid residues [288], with the net charge of the first 53 of them being +18. This region of the protein is likely the one that binds to DNA. At the same time, the carboxyl terminus of this histone exhibits hydrophobic properties and only a minor basic character. Interesting clusters of basic Amino acids have been discovered in certain segments of the polypeptide chain of histone H2a [289]. One of the fascinating Structural Features of histones is the presence of numerous micro-modifications, including phosphorylation of Serine residues, Acetylation and methylation of lysine residues, and methylation of arginine side chains. For example, the Lys-14 and Lys-23 residues in histone H3 are N-acetylated, whereas the Lys-9 and Lys-27 residues are partially ε-N-methylated—each site containing a mixture of partially mono-, di-, and tri-methyl derivatives.

What Functions do histones perform other than neutralizing the charges of DNA? Initially, it was believed that these proteins might act as Gene repressors, much like they do in Bacteria. However, this hypothesis has not received experimental confirmation. Histones appear to form a specific complex with DNA strands. Relatively recently, Electron Cell/15.html">Microscopy has yielded micrographs showing that Chromatin fibers possess a regularly repeating Structure resembling a string of beads. The diameter of the bead (also referred to as a v-body or nucleosome) is 7–10 nm, and the length of the free "string" between beads is 2–14 nm (Fig. 15-35) [290–294]. The DNA content within the "beads" is high. Data obtained from neutron diffraction studies indicate that in v-particles, the DNA strand is wound around a histone oligomer (Fig. 15-36) [295]. Histones H2a, H2b, H3, and H4 are found in nearly equal amounts—approximately one molecule of each histone per every 100 Base Pairs of DNA. An octamer containing two subunits of each histone type has been successfully prepared in solution [296].

Upon nuclease Treatment, chromatin is rapidly cleaved into fragments consisting of 205±15 base pairs, and more slowly into fragments of 170 base pairs. This result, combined with the aforementioned data, suggested the existence of a structure in which a DNA fragment of 200 base pairs is wrapped around a histone octamer in such a way that a double-stranded DNA molecule 68 nm long is packaged into a single v-particle approximately 10 nm in size. Adjacent v-particles are linked to one another by very short stretches of DNA. It has been suggested that the conventional DNA double helix, as it wraps around the histones in a v-particle, may undergo sharp "kinks" every 20 base pairs [297], with the helix unwinding by 15–20° at each such kink. Histone H1, present in lower amounts than the other histones, may act as a cross-linking agent in chromatin (Fig. 15-35). According to other data [296a], there is one negative superhelical turn per v-particle. If this is the case, the number of v-particles in Fig. 15-35 corresponds to the number of superhelical turns in SV40 viral DNA (Fig. 2-27). Histone H1 interacts preferentially with supercoiled DNA [296b]. Cross-linking studies have shown that histone H1 is frequently located in close proximity to several other histones, with histone H2B being equally likely to lie adjacent to H2A, H3, or H4 [298]. As for the chromatin of animals, Fungi, and green plants, its overall structure is fundamentally the same [299].

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FIG. 15-35. A. Electron micrograph of a "minichromosome" formed by SV40 virus grown in cultured monkey cells [292].

In the native form shown, the diameter of the nucleoprotein fiber is approximately 11 nm, and its length is about 210 nm. B. Bead-on-a-string minichromosomes observed upon lowering the Ionic strength. All 21 beads, each about 11 nm in diameter, are connected to one another by bridges approximately 2 nm in diameter and 13 nm in length. Deproteinization and DNA relaxation experiments indicate that the total length of the DNA shown is seven times greater than the total length of the native minichromosome. C. Electron micrograph of a Drosophila melanogaster embryonic chromatid at the blastoderm stage undergoing Replication. Note the presence of nucleosomal (v) particles directly adjacent to the Replication fork (McKnight, L., Miller O. L., Jr.).

Based on the data described above, the prevailing view is that The primary function of histones is to provide the necessary DNA packaging. However, histone H1 is sometimes referred to as a general repressor that maintains chromatin in a compactly packed state, thereby preventing METABOLISM/31.html">Transcription. Because the initiation of mitosis is accompanied by the phosphorylation of histone H1 by a specific protein kinase, it can be hypothesized that this histone plays some other role [300]. Other histones, particularly F4, undergo numerous modifying events, including reversible acetylation and phosphorylation, and irreversible methylation [301]. The Significance of these reactions in regulating processes such as Transcription and Replication remains unclear.

What proteins other than histones are found in cell nuclei? Polyacrylamide gel Electrophoresis has established that HeLa cell nuclei contain about 450 components, most of which are present in small amounts (<10,000 molecules per cell) and are undetectable in the Cytoplasm [302]. The most acidic proteins include A large number of Enzymes, including RNA polymerase. In addition, nuclei contain: 1) specific gene repressors, largely unidentified, 2) hormone-binding proteins, and 3) many other proteins [303]. Alongside nuclear proteins, which usually receive the primary focus, a poorly understood class of Small nuclear RNAs also plays a role in regulating phenotypic Gene Expression. These RNA molecules, ranging from 65 to 200 NUCLEOTIDES in length, can stimulate the transcription of specific genes by binding to complementary DNA sequences. Thus, information transcribed from one region of a chromosome can influence processes occurring at another region or on a different chromosome [303a].

FIG. 15-36. Schematic representation of the putative structure of a chromatin subunit [295].

The protein core is a complex of the non-polar segments of the four histones mentioned in the text. The basic Regions of the histones form a complex with the DNA located On the surface of the nucleosome. Histone H1, positioned between nucleosomes, may act as a cross-linking agent either between nucleosomes of the same strand or between nucleosomes of different strands. The pitch of the DNA helix does not necessarily have to be constant; with an average nucleosome diameter of about 10 nm, it is 5.5 nm.