A)   EUCARYOTIC CHROMOSOMES
            • Size
            • Composition
            • Structure
            • Replication

    B)   ORGANELLE DNA
            • Chloroplast
            • Mitochondria

    C)   REPETITIVE DNA

    D)   SINGLE COPY DNA

    E)   REGULATION OF GENE EXPRESSION
            • DNA-binding proteins again
            (Some old and some new themes)

KEY CONCEPTS:

• Eucaryotic chromosomes (one unique DNA molecule per chromosome!) are larger, have higher degrees of structural order, and a more complex composition than procaryotic chromosomes. The human genome (4x10⁹ bp = 1 meter) is 1,000 times the size of that of E. coli. DNA (4x10⁶ bp = 1.4 mm). Replication and gene expression is more complex than the procaryotic model.

 

• Eucaryotic chromosomal DNA is wound around histones in complexes called nucleosomes. The entire chromosome also contains many other proteins in a complex matrix called chromatin.

 

• A small fraction (2%) of eucaryotic DNA is genes which code for proteins. There is an abundance of repetitive, non-coding sequences which comprise a significant fraction of eucarotic genomes.

 

• Eucaryotic gene expression is regulated primarily at the level of transcription. Transcriptionally active regions of chromosomes are extra-sensitive to DNase digestion and have reduced levels of cytosines which have been methylated. Expression of genes in these chromosomal regions is regulated by transcriptional factors.

 

• A protein (transcription factor IIIA) which regulates 5S rRNA transcription has metal-binding fingers which fit into both major and minor grooves of a B-DNA helix.

 

• Some developmentally-controlled genes in insects and mammals possess a sequence called the homeo box, which codes for a 60 amino acid long DNA-binding polypeptide domain (homeo domain) which may be involved in regulating expression of these genes.

1. SIZE: Large genomes (1 meter long in human genome) linear molecules. [Table 37-1, Fig. 37-2-3]

2. COMPOSITION: Contain five types of basic proteins called histones which have lots (25%) of Arg and Lys residues and are 11 to 21 Kd in mass. The histones are frequently modified by acetylation, methylation, phosphorylation, etc. These modifications may relate to DNA packaging or availability for replication or transcription [Table 37-2]. Histones are highly conserved (especially histones H3 and H4) in all eucaryotes, suggesting that the role of histones was established early during eucaryotic evolution.[Fig. 37-4]

3. STRUCTURE

• Nucleosomes [Fig. 37-5, 8] are repeating units of chromatin which consist of core particles (a histone octamer around which is wrapped 140 base pairs of DNA) connected by linker DNA (20 to 50 base pairs). The core particles contain two molecules each of histones H2A, H2B, H3 and H4 and one molecule of histone H1 binds to the outside of the core particle. The DNA wound around the core particle forms a 1-3/4 turn left-handed superhelix [Fig. 33-8]. Nucleosomes condense DNA [Fig. 37-10] into a smaller space (packing ratio 7:1). Further DNA packing must occur in cells, since metaphase chromosomes have a packing ratio of 1000:1.

• A protein scaffold of non-histone proteins provides a higher order structure and higher packing ratio than that of just nucleosomes. These proteins include topoisomerase II which suggests that changes in supercoiling are important in dynamic changes of in chromosomal DNA structure and function during the cell cycle.

 

• Bidirectional from thousands of replication forks (reduces replication time by orders of magnitude).

• At least three different DNA polymerases ( and are nuclear, is for chromosome DNA replication; gamma is for mitochondrial DNA replication). [Table 37-4]

 

• How to replicate ends of linear DNA chromosomes?? DNA at ends of chromosomes (telomeres) are made by a mechanism [Figure 37-11, Telomerase properties, 37-13, 37-14] using a special telomerase RNA as a template for completing synthesis of the ends of chromosomes.

• New histones form new nucleosomes on the lagging strand while old histones are on the leading strand during replication.

• Eucaryotic cell division cycle [p. 987] has several phases. Mitosis is triggered [Fig. 37-19] by synthesis during the S-phase of cyclin which binds to cdc2-kinase to form an inactive maturation promoting factore (MPF) which is phosphorylated twice. After DNA synthes is completed, one of these phosphates is removed, catalyzed by ccd25 (a phosphatase). This activates the cdc2 kinase activity of the MPF which subsequently phosphorylates many proteins whose activities initiate mitosis. The ccd2 kinase also shuts off its own activity by activating an enzyme which degrades cyclin

 

6. REPETITIVE VS. SINGLE COPY DNA

• Renaturation kinetics (C_ot) analysis shows that eucaryotic DNA has a large fraction of sequences which are repeated (satellites, Alu sequences) up to a million times and a small fraction of unique protein-coding sequences. [Figs. 37-21,22, p.991] Table 37-6 summarizes the several classes of eucaryotic genes.

• Ribosomal RNA genes [Fig . 37-25] are tandemly repeated several hundred times and can be amplified further to 2X10⁶ copies (for instance, during development of Xenopus oocytes) to up to 75% of the total cellular DNA. This amplification provides enough genes for rapid transcription of rRNA to get 10¹² ribosomes/oocyte.

• Histone genes are clustered, repeated, lack introns, and have mRNA which is not polyadenylated. [Fig. 37-27]

 

• Many major (and minor) cell proteins are coded by single copy genes [Figure 37-22, 37-23]. For major cellular proteins, transcription rates, mRNA half-life, and use of mRNA for multiple rounds of translation give up to 10⁹ protein molecules from just one gene copy. However, some single copy genes can also become amplified. For instance, methotrexate can cause amplification of dihydrofolate reductase genes in hamster cell cultures.

 

• Human hemoglobin genes are a family of related genes which are arranged in clusters whose genes have a specific temporal order of expression during development [see Figure 37-28].

 

• Although the human genome is 1000X the size of the E. coli chromosome, only 2% is likely to code for proteins. Thus, the actual unique human genome size is only about 50X that of E. coli. (Does the human genome sequencing project - primary goal of which is to discover all the human genes which code for proteins seem more reasonable now??)

 

• Regions of chromosomes which are transcriptionally active are less condensed (form puffs in Drosophila [Fig. 37-29] are undermethylated, and are hypersensitive to DNase I (mostly at the 5'-ends of genes). These characteristics are tissue-specific and developmentally regulated.

 

• Methylation of cytosines [Fig. 37-30] in chromosomal DNA is associated with low transcriptional activity, in general.

• TRANSCRIPTIONAL ACTIVATORS: We already know about transcription factors and enhancer sequences (see Chapter 33). Transcriptional activators bind to specific activator sequences (enhancers) and these proteins have in common at least two conserved functional domains - one which binds to DNA and one which activates transcription [Fig. 37-31]. In addition, other conserved domains may occur, depending on the nature of the specific type of activator.

 

• Transcription factor proteins are known which possess uo to 37 zinc finger domains. Zinc fingers (9 of them) were discovered in transcription factor IIIA.. They are involved in activating 5S rRNA gene transcription by RNA polymerase III. In a zinc finger domain there are two cysteines and two histidines tetrahedrally-cooridinated with a divalent zinc ion [Fig. 37-32]. These zinc fingers bind to both major and minor grooves of a 50bp internal control region of the 5SrRNA gene and stays bound to the gene during transcription.

• The family of nuclear hormone binding receptors (active as dimers of identical subunits) has an additional ligand-(hormone) binding domain[Figs. 37-35]. The DNA-binding domains of nuclear hormone receptor proteins possess globular structural domains in which four cysteines are tetrahedrally coordinated with a divalent zinc ion[Fig. 37-37]. Two of thesezinc clusters are present on each subunit and they stabilize the structure of of the dimer. Each subunit of the dimeric steroid receptor has an alpha-helix which recognizes and binds to the major groove of the same DNA sequence (its symmetrical) of the steroid response element [Fig. 37-36].

• Another class of eucaryotic transcriptional activators possess DNA-bindingleucine zipper domains. These structural domains are found in proteins which are involved in regulating expression of genes in concert with cyclic AMP. A dimer of the yeast GNC4 protein activates the expression of genes responsible for amino acid biosynthesis after binding to DNA and interacting with transcription factorIID at the TATA box. [Figs. 37-39, 40]

 

• Development in eucaryotes is regulated by groups of genes known as homeotic genes. These genes code for proteins which possess sequences (homeo boxes) which code for polypeptide domains known as homeo domains[Fig. 37-42] of proteins which are developmentally regulated in insects and mammals and which determine body part segmentation and differentiation [Figs. 37-41,44]. Homeo domains have many basic residues and are DNA-binding [Fig.37-43]. Proteins with homeo domains regulate expression of many differentially expressed genes in eucaryotes. Many homeotic genes of Drosophila and the mouse have similar functions and are similarly arranged in clusters on chromosomes [Fig. 37-44]

SUMMARY

· Features of organization and replication of eucaryotic chromosomes and organelle DNA
· Regulation of eucaryotic gene expression (transcription control)
· Regulatory proteins are DNA-binding proteins [DNA binding and gene activation domains]