Eukaryotic Genomes: Organization, Regulation, and Evolution Chapter 19 Eukaryotic Genomes: Organization, Regulation, and Evolution

Overview: How Eukaryotic Genomes Work and Evolve Two features of eukaryotic genomes are a major information-processing challenge: First, the typical eukaryotic genome is much larger than that of a prokaryotic cell Estimated 25,000 genes in the genome Second, cell specialization limits the expression of many genes to specific cells

The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes Part of the chromatin is packed into the main axis, while those parts that are being actively transcribed are spread out in loops Both prokaryotes and eukaryotes must alter their patterns of gene expression in response to changes in environmental conditions

Concept 19.1: Chromatin structure is based on successive levels of DNA packing Eukaryotic DNA is precisely combined with a large amount of protein, and the resulting chromatin undergoes changes in the course of the cell cycle Chromatin—mass within the nucleus Before mitosis, chromatin coils and folds up to form chromosomes Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length Average double helix is 1.5 x 108 nucleotide pairs

Nucleosomes, or “Beads on a String” Proteins called histones are responsible for the first level of DNA packing in chromatin Mass is equal to the mass of DNA High proportion of positively charged amino acids Binds tightly to negatively charged DNA (gets negativity from phosphorus groups) The association of DNA and histones seems to remain intact throughout the cell cycle Animation: DNA Packing

In electron micrographs, unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome, the basic unit of DNA packing Consists of DNA wound around a protein core composed of 2 molecules each of four types of histone “String” is called linker DNA

How can DNA be transcribed when it is wrapped around histones in a nucleosome? Researchers have learned that changes in the shapes and positions of nucleosomes can allow RNA-synthesizing polymerases to move along the DNA

LE 19-2a 2 nm DNA double helix His- Histone tones tails 10 nm Histone H1 10 nm Linker DNA (“string”) Nucleosome (“bead”) Nucleosomes (10-nm fiber)

Higher Levels of DNA Packing The next level of packing forms the 30-nm chromatin fiber Due to interactions between the histone tails of one nucleosome and the linker DNA and nucleosomes to either side

LE 19-2b 30 nm Nucleosome 30-nm fiber

In turn, the 30-nm fiber forms looped domains, making up a 300-nm fiber

LE 19-2c Protein scaffold Loops 300 nm Scaffold Looped domains (300-nm fiber)

In a mitotic chromosome, the looped domains coil and fold, forming the metaphase chromosome

LE 19-2d 700 nm 1,400 nm Metaphase chromosome

Interphase chromatin is usually much less condensed than that of mitotic chromosomes Much of the interphase chromatin is present as a 10-nm fiber, and some is 30-nm fiber, which in some regions is folded into looped domains Although an interphase chromosome lacks a scaffold, its looped domains seem to be attached to the nuclear lamina, on the inside of the nuclear envelope May help organize regions of active transport

Interphase chromosomes have highly condensed areas, called heterochromatin, and less compacted areas, called euchromatin Because of its compaction, heterochromatin DNA is largely inaccessible to transcription enzymes and is not transcribed Looser packing of euchromatin makes its DNA accessible to enzymes and so is not usually transcribed

Concept 19.2: Gene expression can be regulated at any stage, but the key step is transcription All organisms must regulate which genes are expressed at any given time A multicellular organism’s cells undergo cell differentiation, specialization in form and function Human body composed of 200 different cell types

Differential Gene Expression Typical human cell expresses about 20% of all its genes at any given time Differences between cell types result from differential gene expression, the expression of different genes by cells within the same genome In each type of differentiated cell, a unique subset of genes is expressed Many key stages of gene expression can be regulated in eukaryotic cells Most commonly regulated at transcription

Chemical modification Degradation of protein LE 19-3 Signal NUCLEUS Chromatin DNA Gene available for transcription Gene Transcription RNA Exon Primary transcript Intro RNA processing Tail Cap mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein

Regulation of Chromatin Structure Structural organization of chromatin not only packs a cell’s DNA into a compact form that fits inside the nucleus, but also is important in helping regulate gene expression Genes within highly packed heterochromatin are usually not expressed

Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression N terminus end of each histone molecule protrudes outward from the nucleosome Tails are accessible to various modifying enzymes

Histone Modification In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails Deacetylation: removal of acetyl groups When histone tails are acetylated, their positive charges are neutralized and no longer bind to neighboring nucleosomes This process seems to loosen chromatin structure, thereby promoting the initiation of transcription

LE 19-4 Histone tails DNA double helix Amino acids available for chemical modification Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones Acetylation of histone tails promotes loose chromatin structure that permits transcription

DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species DNA that is inactive is generally highly methylated compared with DNA that is actively transcribed In some species, DNA methylation causes long- term inactivation of genes in cellular differentiation Deficient DNA methylation leads to abnormal embryonic development

In genomic imprinting, methylation turns off either the maternal or paternal alleles of certain genes at the start of development

Epigenetic Inheritance Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance Enzymes that modify chromatin structure appear to be important parts of the cell’s machinery for regulating transcription

Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery Once a gene is optimally modified for expression, the initiation of transcription is the most important and universally used state at which gene expression is regulated

Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types

(distal control elements) Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Poly-A signal Primary RNA transcript (pre-mRNA) Exon Intron Exon Intron Exon Cleaved 3¢ end of primary transcript 5¢ RNA processing: Cap and tail added; introns excised and exons spliced together Intron RNA Coding segment mRNA 3¢ Start codon Stop codon 5¢ Cap 5¢ UTR (untranslated region) 3¢ UTR (untranslated region) Poly-A tail

The Roles of Transcription Factors To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

Enhancers and Specific Transcription Factors Proximal control elements are located close to the promoter Distal control elements, groups of which are called enhancers, may be far away from a gene or even in an intron A gene may have multiple enhancers—each active at a different time or in a different cell type or location in the organism Animation: Initiation of Transcription

An activator is a protein that binds to an enhancer and stimulates transcription of a gene Some transcription factors function as repressors, inhibiting expression of a particular gene Some activators and repressors act indirectly by influencing chromatin structure Gene present in region of chromatin with high levels of histone acetylation is able to bind the transcription machinery

Repressors cause inhibition of gene expression in several ways: Block binding of activators Bind directly to their own control elements in an enhancer Turn of transcription in presence of activators

Hundreds of transcription factors have been discovered in eukaryotes Two common structural elements: DNA-binding domain Part of 3D protein shape that binds to DNA Activation domains Bind other regulatory proteins

LE 19-6 Distal control element Activators Promoter Gene DNA Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription Initiation complex RNA synthesis

Combinatorial Control of Gene Activation A particular combination of control elements can activate transcription only when the appropriate activator proteins are present Only about a dozen different nucleotide sequences found in control elements Each enhancer is made of about ten control elements, each of which can bind only one or two specific transcription factors

LE 19-7 Liver cell nucleus Lens cell nucleus Available activators Enhancer Promoter Control elements Albumin gene Albumin gene not expressed Crystallin gene Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed Liver cell Lens cell

Coordinately Controlled Genes How does the prokaryotic cell deal with genes of related function that need to be turned off or on at the same time? Controlled genes often clustered into an operon, which is regulated by a single promoter and transcribed into a single mRNA molecule Genes are expressed together, and encoded proteins are produced at the same time Only found in prokaryotic cells

How does the eukaryotic cell deal with genes of related function that need to be turned off or on at the same time? Research has found that some co-expressed genes are clustered near one another on the same chromosome Each gene has its own promoter and is individually transcribed

Systems for coordinating gene regulation probably arose early in evolutionary history and evolved by the duplication and distribution of control elements within the genome

Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression More and more examples are being found of regulatory mechanisms that operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Animation: RNA Processing RNA processing provide several opportunities for regulating gene expression that are not possible in prokaryotes In alternative RNA splicing, 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 a sequence within the primary transcript Animation: RNA Processing

LE 19-8 Exons DNA Primary RNA transcript RNA splicing or mRNA

mRNA Degradation The life span of mRNA molecules in the cytoplasm is a key to determining the protein synthesis The mRNA life span is determined in part by sequences in the leader and trailer regions Prokaryotic cells: mRNA degraded within a few minutes of their synthesis Short life span allows prokaryotes to quickly change proteins being made in response to environmental changes

Eukaryotic cells: degraded within hours, days, or weeks mRNA breakdown begins with shortening of poly-A tail Then, enzymes remove 5’ cap After, nuclease enzymes rapidly chew up the mRNA Sequences that affect mRNA life span are found in UTR at the 3’ end

Animation: Blocking Translation Animation: mRNA Degradation RNA interference by single-stranded microRNAs (miRNAs) can lead to degradation of an mRNA or block its translation miRNA hydrogen bonds with itself to form double-stranded hairpin structure Enzyme then cuts it into two strands: One strand degraded Other strand binds with protein complex to degrade mRNA or block translation of that mRNA strand Animation: Blocking Translation Animation: mRNA Degradation

The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) Due to small interfering RNAs (siRNAs) Believed that the RNAi pathway originated as a natural defense against infection by RNA viruses

LE 19-9 Protein complex Degradation of mRNA Dicer OR miRNA Target mRNA Hydrogen bond Blockage of translation

Initiation of Translation Translation presents another opportunity for regulating gene expression The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA Found in UTR at the 5’ end Alternatively, translation of all mRNAs in a cell may be regulated simultaneously Involves activation or inactivation of protein factors

Protein Processing and Degradation Final opportunity for controlling gene expression occurs after translation Eukaryotic polypeptides have to be processed to yield functional protein molecules Proteins have to undergo chemical modifications to make them functional Regulatory proteins activated or inactivated Proteins on surface of animal cells require sugar Cell-surface proteins need to be moved Animation: Protein Processing Animation: Protein Degradation

Length of time each protein functions in the cell is strictly regulated by means of selective degradation To mark a protein for destruction, the cell attaches molecules of ubiquitin, a small protein Proteasomes are giant protein complexes that recognize the ubiquitin-tagged protein molecules and degrade them

LE 19-10 Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Protein entering a proteasome