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Eukaryotic Gene Expression Eukaryotic Genomes & Gene Expression Eukaryotic Cells Have Multiple Chromosomes • Eukaryotic cells have 5–20x more DNA per cell than do bacteria. • Divided into linear dsDNA + proteins : chromosomes • Typical chromosome averages ~1.5x108 nucleotide pairs. – If straight strands would be ~ 4 cm long. • Humans have 46 different chromosomes – So collectively amounts to ~ 2 m long dsDNA packed into each cell nucleus! (4 m after replication!) Plant cell just before division Stages in gene expression that can be regulated in eukaryotic cells Chromatin: nondividing cells Signal DNA RNA NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation Gene available for transcription Gene Transcription Exon Primary transcript Intron RNA processing Tail Cap • DNA is coiled to pack into nucleus mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA • Different degrees of coiling and packing Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein Figure 18.6 Levels of chromatin packing Histone tails and the effect of acetylation 1. Transcription factors may catalyze histone acetylation 2. Acetylated histone tails may recruit transcription factors 2 nm DNA double helix Histones Histone tails Histone H1 Beads of eight histone proteins 10 nm DNA double helix Linker DNA (“string”) Nucleosome (“bead”) chromatin (a) Nucleosomes (10-nm fiber) 30 nm Amino acids on tails available for chemical modification (a) Histone tails protrude outward from a nucleosome Dense regions of heterochromatin Nucleosome Unacetylated histones DNA remains coiled around histone “beads” except during replication. Figure 18.7 Heyer Histone tails Less dense regions of euchromatin Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Figure 18.7 1 Eukaryotic Gene Expression DNA in a eukaryotic chromosome from a developing salamander egg Chromosomes in the interphase nucleus Chromosome territory 10 µm Chromatin loop Transcription factory Figure 18.12 More loosely coiled loops are available for transcription and gene expression. During cell division Levels of chromatin packing 30 nm Nucleosome chromatin (b) 30-nm fiber Protein scaffold Loops 300 nm Scaffold (c) Looped domains (300-nm fiber) • chromatin condenses into visible chromosomes 700 nm chromosome • Tightly wound chromosomes segregate without tangling together 1,400 nm (d) Metaphase chromosome Cell Differentiation in Multicellular Organisms Differential Gene Expression • Even though every cell has the same genome, each cell type only uses a small subset of genes. – – – – ~200 cell types in mammals Each uses only ~20% of total genes Fewer in more specialized cells Unused genes may be permanently inactivated Processing of “primary transcript” RNA [Review Gene Expression slides!] • Histone modification 5ʹ′ – Methylation of histone residues may condense associated DNA into non-transcribable heterochromatin • DNA methylation – Methylation of cytosines related to gene inactivation – Methylated DNA may attract/bind histone deacetylation enzymes – Epigenic inheritance — patterns of methylation passed on to daughter cells Figure 47.7 Blastulation Heyer 50 to 250 adenine nucleotides added to the 3ʹ′ end by A modified methyl-guanine nucleotide added to the 5ʹ′ end poly-A synthetase using ATPs Protein-coding segment G P P P 5ʹ′ Cap Polyadenylation signal AAUAAA 5ʹ′ UTR Start codon Stop codon 3ʹ′ UTR 3ʹ′ AAA…AAA Poly-A tail • Cap & tail protect mRNA from rapid degradation in the cytoplasm. • Eukaryotic mRNA stay active for hours, or even days, in the cytoplasm. • Prokaryotes lack cap & tail; mRNA only lasts for minutes. Figure 17.9 2 Eukaryotic Gene Expression A eukaryotic gene and its transcript Enhancer Proximal (distal control elements) control elements A model for the action of enhancers and transcription activators Distal control element Poly-A signal Termination sequence region Exon Intron Downstream Poly-A signal Cleared 3ʹ′ end Intron Exon of primary RNA processing: transport Cap and tail added; introns excised and exons spliced together Transcription Promoter Primary RNA transcript 5ʹ′ (pre-mRNA) Chromatin changes Exon Intron Exon Transcription Intron RNA RNA processing mRNA degradation TATA box 1 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. General transcription factors DNA-bending protein 2 A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. Coding segment Translation Protein processing and degradation Gene Enhancer DNA Upstream Promoter Activators Intron Exon Exon Group of Mediator proteins RNA Polymerase II mRNA G P P P 5ʹ′ Cap 5ʹ′ UTR (untranslated region) Figure 18.8 Start codon Stop codon 3ʹ′ UTR Poly-A tail (untranslated region) • Enhancer sequences may be several kb upstream or downstream of the gene, or within an intron. • One gene may have several enhancers. Chromatin changes 3 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. Transcription RNA processing mRNA degradation RNA Polymerase II Translation Protein processing and degradation Transcription Initiation complex RNA synthesis Figure 18.10 Cell type–specific transcription An Activator Protein and DNA-binding Promoter Enhancer Albumin gene Control elements Crystallin gene Lens cell nucleus Liver cell nucleus Activation domain DNA-binding domain Available activators Available activators DNA Albumin gene not expressed Albumin gene expressed Crystallin gene Crystallin gene not expressed expressed (a) Liver cell (b) Lens cell Figure 18.9 Sequential Regulation of Gene Expression During Cellular Differentiation Nucleus Embryonic precursor cell Master regulatory gene myoD Other muscle-specific genes Alternative RNA splicing • Esp. in vertebrate animals • >1 possible polypeptide/gene! DNA OFF OFF Figure 18.11 Chromatin changes Transcription RNA processing OFF mRNA Myoblast (determined) mRNA degradation MyoD protein (transcription factor) Translation Protein processing and degradation Exons DNA mRNA Part of a muscle fiber (fully differentiated cell) Heyer MyoD mRNA Another transcription factor mRNA mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins Figure 18.18 Primary RNA transcript RNA splicing or mRNA Figure 18.13 3 Eukaryotic Gene Expression Regulation of gene expression by microRNAs (miRNAs) 1 The microRNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 2 An enzyme called Dicer moves along the doublestranded RNA, cutting it into shorter segments. 3 One strand of each short doublestranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 4 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. Degradation of a protein by a proteasome 5 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. Chromatin changes Transcription Chromatin changes 1 Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol. 2 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. Transcription RNA processing RNA processing mRNA degradation Translation Protein processing and degradation Protein complex mRNA degradation Proteasome and ubiquitin to be recycled Ubiquitin Translation Proteasome Protein processing and degradation Dicer Degradation of mRNA OR miRNA Protein to be degraded Ubiquinated protein Protein entering a proteasome Target mRNA Protein fragments (peptides) Blockage of translation Hydrogen bond Figure 18.14 See Figure 18.15 Movement of eukaryotic transposable elements The effect of transposable elements on corn kernel color New copy of transposon Transposon DNA of genome Transposon is copied Insertion Mobile transposon (a) Transposon movement (“copy-and-paste” mechanism) New copy of Retrotransposon retrotransposon Figure 21.9 DNA of genome RNA Reverse transcriptase Insertion (b) Retrotransposon movement Figure 21.10 Types of DNA sequences in the human genome Exons (1.5%) Alu elements (10%) Encode Project: Rethinking ‘Junk’ DNA Introns (5%) GENES Each human cell contains about 10 feet of DNA, coiled into a dense tangle. But only a very small percentage of DNA encodes genes, which control inherited traits like eye color, blood type and so on Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (14%) Simple sequence DNA (3%) Heyer Figure 21.8 Regulatory sequences (∼20%) Repetitive DNA that includes transposable elements and related sequences (44%) L1 sequences (17%) Barbara McClintock’s breeding experiments with Indian corn Large-segment duplications (5-6%) Figure 21.7 “JUNK” DNA [“DARK MATTER”] Stretches of DNA around and between genes seemed to do nothing, and were called junk DNA. But now researchers think that the junk DNA contains a large number of *ny gene*c switches, controlling how genes func*on within the cell. Over half of the human genome is gene switches. REGULATION DISEASE The many gene*c regulators seem to be arranged in a complex and redundant hierarchy. Scien*sts are only beginning to map and understand this network, which regulates how cells, organs and *ssues behave. Errors or muta*ons in gene*c switches can disrupt the network and lead to a range of diseases. The new findings will spur further research and may lead to new drugs and treatments. Bits of Mystery DNA, Far From ‘Junk,’ Play Crucial Role By GINA KOLATA; September 5, 2012, NY Times National Human Genome Research Institute Encyclopedia Of DNA Elements 4