The Organization and Control of Eukaryotic Genomes Chapter 19

REMEMBER: Prokaryotic Genomes http://highered. mcgraw-hill shape (circular/nonlinear/loop) less complex than eukaryotes (no histones/less elaborate structure/folding) size (smaller size/less genetic information/fewer genes) replication method (single origin of replication/rolling circle replication) transcription/translation may be coupled generally few or no introns (noncoding segments) majority of genome expressed operons are used for gene regulation and control NOTE: plasmids – more common but not unique to prokaryotes/not part of prokaryote chromosome

Overview Figure 19.7 Opportunities for the control of gene expression in eukaryotes: Chromatin Packing, modification Assembling of Transcription Factors RNA Processing Regulation of mRNA degradation and Control of Translation Protein Processing and Degradation THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE. The expression of a given gene will not necessarily involve every stage shown. MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down.

Expression of Genes Eukaryotic cells face the same challenges as prokaryotic cells in expressing their genes, but with two main differences: The much greater size of the typical eukaryotic genome; importance of cell specialization in multicellular eukaryotes. In both prokaryotes and eukaryotes, DNA associates with proteins to form chromatin, but in the eukaryotic cell, the chromatin is ordered into higher structural levels.

Eukaryotic Chromosome Structure Chromatin structure is based on successive levels of DNA packing. Eukaryotic chromatin is composed mostly of DNA and histone proteins that bind to the DNA to form nucleosomes, the most basic units of DNA packing. Additional folding leads ultimately to highly compacted heterochromatin, the form of chromatin in a metaphase chromosome. In interphase cells, most chromatin is in a highly extended form, called euchromatin. Chromatin structure is based on successive levels of DNA packing. Chromatin = term for DNA + Proteins in the Nucleus of a Eukaryotic cell DNA is wound around Histones (positively charged proteins which attract negatively charged DNA) Histones (8) with wound DNA make up a Nucleosome - the fundamental packing unit of Chromosome (10nm fiber) Nucleosomes further condense and form 30nm Chromatin Fibers Nucleosomes scaffold, loop, fold to make Looped Domains (300nm – 700nm fibers) Metaphase Chromosome – most condensed, largest form of DNA (1400nm) 5

The Eukaryotic Genome In prokaryotes, most of the DNA in a genome codes for protein, with a small amount of noncoding DNA that consists mainly of regulatory sequences such as promoters. In eukaryotic genomes, most of the DNA (97% in humans) does NOT encode protein or RNA. This DNA includes introns and repetitive DNA: Repetitive DNA are nucleotide sequences that are present in many copies in a genome, usually not within genes.

Gene Amplification, Loss, or Rearrangement Gene amplification, loss, or rearrangement can alter a cell’s genome during an organisms lifetime: Sometimes the number of copies of a gene or gene family temporarily increases in the cells of some tissues during a particular stage of development: Known as gene amplification – a potent way of increasing expression of the genes enabling a developing egg to make enormous numbers of ribosomes. Genes can also be selectively lost in certain tissues (seen in early insect development).

Rearrangement in the Genome All organisms seem to have transposons: These can increase or decrease the production of one or more proteins depending on where the sequence inserts (jumps).

Cellular Differentiation Gene expression must be controlled on a long-term basis for cellular differentiation during an organism’s development. In all organisms, the expression of specific genes is most commonly regulated at the level of transcription by DNA-binding proteins. For this reason, the term gene expression is often equated with gene activity – transcription – for both prokaryotes and eukaryotes. However, the greater complexity of eukaryotic cell structure and function provides opportunities for controlling gene expression at additional stages.

Overview Figure 19.7 Opportunities for the control of gene expression in eukaryotes: Chromatin Packing, modification Assembling of Transcription Factors RNA Processing Regulation of mRNA degradation and Control of Translation Protein Processing and Degradation THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE. The expression of a given gene will not necessarily involve every stage shown. MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down. This figure highlights key stages in the expression of a protein-coding gene, from chromatin changes that unpack the DNA, through transcription, RNA processing, and translation, to various alterations of the protein produce. Each stage is a potential point where gene expression can be turned on or off, speeded up, or slowed down.

Chromatin Modifications Chromatin modifications affect the availability of genes for transcription: The physical state of DNA in or near a gene is important in helping control whether the gene is available for transcription. Genes of heterochromatin (highly condensed) are usually not expressed because transcription proteins cannot reach the DNA. DNA methylation seems to diminish transcription of that DNA. Histone acetylation seems to loosen nucleosome structure and thereby enhance transcription.

DNA Methylation DNA methylation is the attachment of methyl groups (-CH3) to DNA bases after DNA is synthesized. Inactive DNA, such as that of inactivated mammalian X chromosomes, is generally highly methylated compared to DNA that is actively transcribed. Comparison of the same genes in different types of tissues shows that the genes are usually more heavily methylated in cells where they are not expressed. In addition, demethylating certain inactive genes (removing their extra methyl groups) turns them on. At least in some species, DNA methylation seems to be essential for the long-term inactivation of genes that occurs during cellular differentiation in the embryo.

Histone Acetylation Histone acetylation is the attachment of acetyl groups (-COCH3) to certain amino acids of histone proteins; deacetylation is the removal of acetyl groups. When the histones of nucleosome are acetylated, they change shape so that they grip the DNA less tightly. As a result, transcription proteins have easier access to genes in the acetylated region.

Transcription Initiation Transcription initiation is controlled by proteins that interact with DNA and with each other. Once a gene is “unpacked”, the initiation of transcription is the most important and universally used control point in gene expression.

Eukaryotic Gene and its Transcript – Figure 19.8

Assembling of Transcription Factors Activator proteins bind to enhancer sequences in the DNA and help position the initiation complex on the promoter. DNA bending brings the bound activators closer to the promoter. Other transcription factors and RNA polymerase are nearby. Protein-binding domains on the activators attach to certain transcription factors and help them form an active transcription initiation complex on the promoter. http://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf Control elements are simply segments of noncoding DNA that help regulate transcription of a gene by binding proteins (transcription factors). 16

Post-Transcriptional Factors Transcription alone DOES NOT constitute gene expression! Post-transcriptional mechanisms play supporting roles in the control of gene expression: Alternative RNA splicing – where different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript. http://highered.mcgraw-hill.com/olc/dl/120080/bio31.swf

Alternative Splicing Offers New Combinations of Exons = New Proteins The RNA transcripts of some genes can be spliced in more than one way, generating different mRNA molecules. With alternative splicing, an organism can get more than one type of polypeptide from a single gene. 18

Further Control of Gene Expression After RNA processing, other stages of gene expression that the cell may regulate are mRNA degradation, translation initiation, and protein processing and degradation. The life span of mRNA molecules in the cytoplasm is an important factor in determining the pattern of protein synthesis in a cell. Most translational control mechanisms block the initiation stage of polypeptide synthesis, when ribosomal subunits and the initiator tRNA attach to an mRNA.

Protein Processing and Degradation The final opportunities for controlling gene expression occur after translation: Protein processing – cleavage and the addition of chemical groups required for function. Transport of the polypeptide to targeted destinations in the cell. Cells can also limit the lifetimes of normal proteins by selective degradation – chopped up by proteasomes.

The Molecular Biology of Cancer Certain genes normally regulate growth and division – the cell cycle – and mutations that alter those genes in somatic cells can lead to cancer. Proto-Oncogenes are normal genes that code for proteins which stimulate normal cell growth and division. Oncogenes – cancer causing genes; lead to abnormal stimulation of cell cycle. Oncogenes arise from a genetic changes in proto-oncogene Amplification of proto-oncogenes Point mutation in proto-oncogene Movement of DNA within genome

Genetic Changes Can Turn Proto-oncogenes into Oncogenes http://www 22

Tumor-Suppressor Genes In addition to mutations affecting growth-stimulating proteins, changes in genes whose normal products INHIBIT cell division also contribute to cancer: Such genes are called tumor-suppressor genes because the proteins they encode normally help prevent uncontrolled cell growth.

p53 Tumor Suppressor and ras Proto-Oncogenes http://www. learner Mutations in the p53 tumor-suppressor gene and the ras proto-oncogene are very common in human cancers. Both are components of signal-transduction pathways that convey external signal to the DNA in the cell’s nucleus. Product of ras gene is G Protein (relays a growth signal and stimulates cell cycle). An oncogene protein that is a hyperactive version of this protein in the pathway can increase cell division. P53 protein – “guardian angel of the genome” DNA damage (UV, toxins) signals expression of p53 and p53 protein acts as transcription factor for gene p21 p21 halts cell cycle, allowing DNA repair P53 also can cause ‘cell suicide’ if damage is too great Many cancer patients p53 gene product does not function properly!

Figure 19.14 Signaling pathways that regulate cell growth (Layer 2) RAS and P53 contribute to uninhibited cell stimulation and growth- Tumor Formation 25

Figure 19.15 A multi-step model for the development of colorectal cancer 26

Review: Structure/Function of Eukaryotic Chromosomes Chromatids 2/sister/pari/identical DNA/ genetic information distribution of one copy to each new cell Centromere noncoding/uncoiled/narrow/constricted region joins/holds/attaches chromatids together Nucelosome histones/DNA wrapped arround special proteins packaging compacting Chromatin Form (heterochromatin/euchromatin) heterochromatin is condensed/supercoiled proper distribution in cell division (not during replication) euchromatin is loosely coiled gene expression during interphase/replication occurs when loosely packed Kinetochores disc-shaped proteins spindle attachment/alignment Genes or DNA brief DNA description codes for proteins or for RNA Telomeres tips, ends, noncoding repetitive sequences protection against degradation/ aging, limits number of cell divisions

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