Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 - can impact efficiency of gene expression – Second, cell specialization limits the expression of many genes to specific cells

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 19.1: Chromatin structure is based on successive levels of DNA packing Eukaryotic DNA is precisely combined with a large amount of protein The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA- protein complex in prokaryotes Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Nucleosomes, or “Beads on a String” Proteins called histones are responsible for the first level of DNA packing in chromatin The association of DNA and histones seems to remain intact throughout the cell cycle In electron micrographs, unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome, the basic unit of DNA packing

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Higher Levels of DNA Packing The next level of packing forms the 30-nm chromatin fiber Interactions between histone tails, linker DNA, and other nucleosomes cause the 10-nm fiber to coil and fold, forming a chromatin fiber approximately 30 nm in diameter

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In turn, the 30-nm fiber forms looped domains, making up a 300-nm fiber During prophase of mitosis or meiosis, the 30-nm fiber forms looped domains by attaching to a non- histone protein scaffold, giving rise to a 300-nm fiber

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In a mitotic chromosome, the 300-nm looped domains coil and fold, forming the metaphase chromosome

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Interphase chromosomes have highly condensed areas, called heterochromatin, and less compacted areas, called euchromatin Animation: DNA Packing Animation: DNA Packing

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 which is a specialization in form and function

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Differential Gene Expression Differences between cell types result from differential gene expression, the expression of different genes by cells possessing 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

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Regulation of Chromatin Structure Genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Histone Modification In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species In some species, DNA methylation causes long- term inactivation of genes in cellular differentiation

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Control elements are loosely analogous (similar in concept) to the operator of the prokaryotic operon in that binding of certain factors will influence the levels of transcription

LE 19-5 Enhancer (distal control elements) Proximal control elements Upstream DNA Promoter ExonIntron ExonIntron Exon Downstream Transcription Poly-A signal sequence Termination region Intron ExonIntron Exon RNA processing: Cap and tail added; introns excised and exons spliced together Poly-A signal Cleaved 3 end of primary transcript 3 Poly-A tail 3 UTR (untranslated region) 5 UTR (untranslated region) Start codon Stop codon Coding segment Intron RNA 5 Cap mRNA Primary RNA transcript (pre-mRNA) 5 Exon

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 An activator is a protein that binds to an enhancer and stimulates transcription of a gene Animation: Initiation of Transcription Animation: Initiation of Transcription

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Some transcription factors function as repressors, inhibiting expression of a particular gene Some activators and repressors act indirectly by influencing chromatin structure - recruiting proteins that will affect levels of histone acetylation: Will activators tend to recruit proteins that acetylate or de-acetylate histone? What about repressors?

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Combinatorial Control of Gene Activation A particular combination of control elements can activate transcription only when the appropriate activator proteins are present

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Coordinately Controlled Genes Unlike the genes of a prokaryotic operon, coordinately controlled eukaryotic genes each have a promoter and control elements The same regulatory sequences are common to all the genes of a group, enabling recognition by the same specific transcription factors

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 post-transcriptional mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings RNA Processing 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 Animation: RNA Processing Animation: RNA Processing

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Initiation of Translation The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA, usually found in either the 5´ or 3´ UTR Additionally, a poly-A tail of insufficient length can inhibit efficient translation of a transcript Alternatively, translation of all mRNAs in a cell may be regulated simultaneously by mass activation or inactivation of translation initiation factors

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups (such as phosphate groups), are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them Animation: Protein Processing Animation: Protein Processing

LE Protein to be degraded Ubiquitinated protein Proteasome Protein entering a proteasome Protein fragments (peptides) Proteasome and ubiquitin to be recycled Ubiquitin Animation: Protein Degradation Animation: Protein Degradation