14. The Eukaryotic Genome and Its Expression

The Eukaryotic Genome and Its Expression14 The Eukaryotic Genome and Its Expression14.1 What Are the Characteristics of the Eukaryotic Genome?14.2 What Are the Characteristics of Eukaryotic Genes?14.3 How Are Eukaryotic Gene Transcripts Processed?14.4 How Is Eukaryotic Gene Transcription Regulated?14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?14.6 How Is Gene Expression Controlled During and After Translation?14.1 What Are the Characteristics of the Eukaryotic Genome?Key differences between eukaryotic and prokaryotic genomes:Eukaryotic genomes are larger.Eukaryotic genomes have more regulatory sequences.Much of eukaryotic DNA is noncoding. 14.1 What Are the Characteristics of the Eukaryotic Genome?Eukaryotes have multiple chromosomes.In eukaryotes, translation and transcription are physically separated which allows many points of regulation before translation begins.Figure 14.1 Eukaryotic mRNA is Transcribed in the Nucleus but Translated in the CytoplasmTable 14.114.1 What Are the Characteristics of the Eukaryotic Genome?Eukaryote model organisms:Yeast, Saccharomyces cerevisiaeNematode (roundworm), Caenorhabditis elegansFruit fly, Drosophila melanogasterThale cress, Arabidopsis thaliana14.1 What Are the Characteristics of the Eukaryotic Genome?The yeast (Saccharomyces cerevisiae) has 16 chromosomes; haploid content of 12 million base pairs (bp).Compartmentalization into organelles requires more genes than prokaryotes have.Table 14.214.1 What Are the Characteristics of the Eukaryotic Genome?Some eukaryotic genes that have no homologs in prokaryotes:Genes encoding histonesGenes encoding cyclin-dependent kinases that control cell divisionGenes encoding proteins involved in processing of mRNA14.1 What Are the Characteristics of the Eukaryotic Genome?The soil nematode, Caenorhabditis elegans, is only 1 mm long.A model organism to study development: the body is transparent, an adult has about 1,000 cellsThe genome is eight times larger than yeasts.Table 14.314.1 What Are the Characteristics of the Eukaryotic Genome?Drosophila melanogaster has been used extensively in genetic studies.Genome is larger than C. elegans, but has fewer genesThe genome codes for more proteins than it has genes.Figure 14.2 Functions of the Eukaryotic Genome14.1 What Are the Characteristics of the Eukaryotic Genome?Arabidopsis thaliana is in the mustard family.Has some genes that have homologs in C. elegans and DrosophilaAlso has genes that distinguish it as a plant, such as genes for photosynthesis.Table 14.414.1 What Are the Characteristics of the Eukaryotic Genome?Rice (Oryza sativa) genome has also been sequenced—two subspeciesHas many genes similar to Arabidopsis.Table 14.514.1 What Are the Characteristics of the Eukaryotic Genome?Eukaryote genomes have two types of highly repetitive sequences that do not code for proteins:Minisatellites: 10–40 bp, repeated several thousand times. Number of copies varies among individuals—provides molecular markers.Microsatellites: 1–3 bp, 15–100 copies14.1 What Are the Characteristics of the Eukaryotic Genome?Moderately repetitive sequences (genes): code for tRNA and rRNAThese molecules are needed in large quantities; the genome has multiple copies of the sequence.14.1 What Are the Characteristics of the Eukaryotic Genome?Mammals: Four different rRNAs16S, 5.8S, 28S are transcribed as a single precursor molecule. Humans have 280 copies of the sequence on five different chromosomes; and 5S.(S = Svedberg unit)Figure 14.3 A Moderately Repetitive Sequence Codes for rRNA14.1 What Are the Characteristics of the Eukaryotic Genome?Other moderately repetitive sequences can move from place to place in the genome—transposons.Transposons make up 40 percent of human genome, only 3–10 percent in other sequenced eukaryotes.14.1 What Are the Characteristics of the Eukaryotic Genome?Four types of transposons:SINEsLINEsRetrotransposonsDNA transposons14.1 What Are the Characteristics of the Eukaryotic Genome?SINEs (short interspersed elements)—500 bp; 15 percent of human DNA. One, Alu, is present in a million copiesLINEs (long interspersed elements)—7,000 bp; about 17 percent of human DNA; some code for proteins14.1 What Are the Characteristics of the Eukaryotic Genome?SINEs and LINEs make an RNA copy of themselves that is a template for new DNA inserted somewhere else—“copy and paste” mechanism.14.1 What Are the Characteristics of the Eukaryotic Genome?Retrotransposons: about 8 percent of human genome; also make an RNA copy of themselves.DNA transposons move to a new place in the genome without replicating.Figure 14.4 DNA Transposons and Transposition14.1 What Are the Characteristics of the Eukaryotic Genome?The function of the transposons is unclear.They may be cellular parasites.If a transposon is inserted into a coding region, a mutation results. If it’s in a somatic cell, cancer can result. Transposons can carry genes to new locations—adding to genetic variation.14.1 What Are the Characteristics of the Eukaryotic Genome?Transposons may have played a role in endosymbiosis:Genes from the once-independent prokaryotes may have moved to the nucleus by DNA transposons.14.2 What Are the Characteristics of Eukaryotic Genes?Gene characteristics not found in prokaryotes:Eukaryote genes contain noncoding internal sequences.Form gene families—groups of structurally and functionally related genes14.2 What Are the Characteristics of Eukaryotic Genes?Eukaryote genes have a promoter to which RNA polymerase binds and a terminator sequence to signal end of transcription.Terminator sequence comes after the stop codon.Stop codon is transcribed into mRNA and signals the end of translation at the ribosome.Figure 14.5 Transcription of a Eukaryotic Gene (Part 1)Figure 14.5 Transcription of a Eukaryotic Gene (Part 2)14.2 What Are the Characteristics of Eukaryotic Genes?Protein-coding genes have noncoding sequences—introns.The coding sequences are extrons.Transcripts of introns appear in the pre-mRNA, they are removed from the final mRNA.14.2 What Are the Characteristics of Eukaryotic Genes?Nucleic acid hybridization reveals introns.Target DNA is denatured; then incubated with a probe—a nucleic acid strand from another source.If the probe has a complementary sequence, base pairing forms a hybrid.Figure 14.6 Nucleic Acid Hybridization14.2 What Are the Characteristics of Eukaryotic Genes?If researchers used mature mRNA as the probe, the DNA-RNA hybrid would have loops where base pairing did not occur—the introns.If pre-mRNA was used, resulted in complete hybridizationFigure 14.7 Nucleic Acid Hybridization Revealed the Existence of Introns (Part 1)Figure 14.7 Nucleic Acid Hybridization Revealed the Existence of Introns (Part 2)14.2 What Are the Characteristics of Eukaryotic Genes?Introns interrupt, but do not scramble, the DNA sequence that encodes a polypeptide.Sometimes, the separated exons code for different domains (functional regions) of the protein.14.2 What Are the Characteristics of Eukaryotic Genes?About half of the eukaryote genes are present in multiple copies.Different mutations can occur in copies, giving rise to gene families.Family that encodes for immunoglobulins have hundreds of members.14.2 What Are the Characteristics of Eukaryotic Genes?As long as one member of a gene family retains the original sequence, copies can mutate without losing original function.This is important in evolution.14.2 What Are the Characteristics of Eukaryotic Genes?The globin gene family arose from a common ancestor gene.In humans:Alpha-globin (α-globin)—three functional genesBeta-globin (β-globin)—five functional genesHemoglobin is a tetramer of two α units and two β units.Figure 14.8 The Globin Gene FamilyFigure 3.9 Quaternary Structure of a Protein14.2 What Are the Characteristics of Eukaryotic Genes?During development, different globin genes are expressed at different times: differential gene expression.γ-globin is in hemoglobin of human fetus—it binds oxygen more tightly than adult hemoglobin.Figure 14.9 Differential Expression in the Globin Gene Family14.2 What Are the Characteristics of Eukaryotic Genes?Some gene families have pseudogenes—result from a mutation that results in loss of function.Pseudogenes may lack a promoter, or recognition sites for removal of introns.Designated by ψ (psi)14.3 How Are Eukaryotic Gene Transcripts Processed?In the nucleus, pre-mRNA is modified at both ends:G-cap added at the 5′ end (modified guanosine triphosphate)—facilitates binding to ribosome.Protects it from being digested by ribonucleases.14.3 How Are Eukaryotic Gene Transcripts Processed?Poly A tail added at 3′ end.AAUAAA sequence after last codon is a signal for an enzyme to cut the pre-mRNA; then another enzyme adds 100 to 300 adenines—the “tail.”May assist in export from nucleus; important for stability of mRNA.Figure 14.10 Processing the Ends of Eukaryotic Pre-mRNA (Part 1)Figure 14.10 Processing the Ends of Eukaryotic Pre-mRNA (Part 2)14.3 How Are Eukaryotic Gene Transcripts Processed?RNA splicing removes introns and splices exons together.Pre-mRNA is bound by small nuclear ribonucleoprotein particles (snRNPs).Consensus sequences are short sequences between exons and introns. snRNP binds here, and also near the 3′ end of the intron.14.3 How Are Eukaryotic Gene Transcripts Processed?With energy from ATP, proteins are added to form an RNA-protein complex, the spliceosome.The complex cuts pre-mRNA, releases introns, and splices exons together.Figure 14.11 The Spliceosome: An RNA Splicing Machine14.3 How Are Eukaryotic Gene Transcripts Processed?In the disease beta thalassemia, a mutation occurs at the consensus sequence in the β-globin gene—the pre-mRNA can not be spliced correctly.Non-functional β-globin mRNA is produced.14.3 How Are Eukaryotic Gene Transcripts Processed?Mature mRNA leaves the nucleus through nuclear pores.TAP protein binds to the 5′ end, TAP binds to other proteins that are recognized by receptors at the nuclear pore.14.4 How Is Eukaryotic Gene Transcription Regulated?Expression of genes must be precisely regulated during development.Gene expression can be regulated at several points in the transcription and translation processes.Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 1)Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 2)Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 3)14.4 How Is Eukaryotic Gene Transcription Regulated?Transcriptional regulation and posttranscriptional regulation can be determined by examining mRNA sequences made in different cell types.14.4 How Is Eukaryotic Gene Transcription Regulated?Eukaryote genes are not organized into operons.Regulation of several genes at once requires common control elements.Eukaryotes have three RNA polymerases:I codes for rRNA; III codes for tRNAII transcribes protein-coding genes14.4 How Is Eukaryotic Gene Transcription Regulated?Most eukaryotic genes have sequences that regulate rate of transcription.Initiation of transcription involves many proteins (in contrast to prokaryotes in which RNA polymerase directly recognized the promoter).14.4 How Is Eukaryotic Gene Transcription Regulated?In prokaryotes, promoter has two sequences:The recognition sequence is recognized by RNA polymerase.The TATA box, where DNA begins to denature.14.4 How Is Eukaryotic Gene Transcription Regulated?In eukaryotes, transcription factors (regulatory proteins) must assemble on the chromosome before RNA polymerase can bind to the promoter.TFIID binds to the TATA box; then other transcription factors bind, forming a transcription complex.Figure 14.13 The Initiation of Transcription in Eukaryotes (Part 1)Figure 14.13 The Initiation of Transcription in Eukaryotes (Part 2)14.4 How Is Eukaryotic Gene Transcription Regulated?Some sequences are common to promoters of many genes; recognized by transcription factors in all cells.Some sequences are specific to a few genes and are recognized by transcription factors found only in certain tissues. These play an important role in differentiation.14.4 How Is Eukaryotic Gene Transcription Regulated?Regulator sequences are located upstream of the promoter.Regulator proteins bind to these sequences. Resulting complex binds to the transcription complex to activate transcription.14.4 How Is Eukaryotic Gene Transcription Regulated?Enhancer sequences are farther away—up to 20,000 bp.Activator proteins bind to enhancer sequences, which stimulates transcription complex. Mechanism not known; perhaps by DNA bending.Figure 14.14 Transcription Factors, Regulators, and Activators (Part 1)Figure 14.14 Transcription Factors, Regulators, and Activators (Part 2)14.4 How Is Eukaryotic Gene Transcription Regulated?Negative regulatory sequences or silencer sequences turn off transcription by binding repressor proteins.14.4 How Is Eukaryotic Gene Transcription Regulated?DNA-binding proteins have four structural themes or motifs:Helix-turn-helixZinc fingerLeucine zipperHelix-loop-helixFigure 14.15 Protein–DNA Interactions (Part 1)Figure 14.15 Protein–DNA Interactions (Part 2)Figure 14.15 Protein–DNA Interactions (Part 3)Figure 14.15 Protein–DNA Interactions (Part 4)14.4 How Is Eukaryotic Gene Transcription Regulated?Bases in DNA can form hydrogen bonds with proteins, especially in major and minor grooves.Many repressor proteins have helix-turn-helix configuration—binding of repressor prevents other proteins from binding and initiating transcription.14.4 How Is Eukaryotic Gene Transcription Regulated?Regulation of genes that are far apart or on different chromosomes—genes must have same regulator sequences.Example: Some plant genes have a regulatory sequence called stress response element (SRE).Genes with this sequence encode for proteins needed to cope with drought.Figure 14.16 Coordinating Gene Expression (Part 1)Figure 14.16 Coordinating Gene Expression (Part 2)14.4 How Is Eukaryotic Gene Transcription Regulated?Transcription can also be regulated by changes in chromatin and chromosomes.14.4 How Is Eukaryotic Gene Transcription Regulated?Chromatin remodeling:DNA is wound around histones to form nucleosomes, which block initiation and elongation.One remodeling protein disaggregates the nucleosome to allow initiation.The second remodeling protein binds to the nucleosomes to allow elongation to proceed.Figure 14.17 Local Remodeling of Chromatin for Transcription (Part 1)Figure 14.17 Local Remodeling of Chromatin for Transcription (Part 2)14.4 How Is Eukaryotic Gene Transcription Regulated?Histone proteins have “tails” with positively charged amino acids—enzymes add acetyl groups:14.4 How Is Eukaryotic Gene Transcription Regulated?This reduces positive charges, and decreases affinity of histones for negatively charged DNA.Allows chromatin remodeling14.4 How Is Eukaryotic Gene Transcription Regulated?Gene activation requires histone acetyl transferases to add acetyl groups.Gene repression requires histone deacetylases to remove the acetyl groups.14.4 How Is Eukaryotic Gene Transcription Regulated?The “histone code”—histone modifications affect gene activation and repression.Example: Methylation of histones is associated with gene inactivation.Whether a gene becomes activated by chromatin remodeling may be determined by histone modification.14.4 How Is Eukaryotic Gene Transcription Regulated?Two types of chromatin:Euchromatin contains DNA that is transcribed into mRNA.Heterochromatin: genes it contains are usually not transcribed. 14.4 How Is Eukaryotic Gene Transcription Regulated?Example of heterochromatin: inactive X chromosome in mammals. Each female has two copies of genes on the X chromosome.Y chromosome gradually lost most of the genes it once shared with its X homolog.Female has potential to produce twice as much protein from the X-linked genes.14.4 How Is Eukaryotic Gene Transcription Regulated?One X chromosome remains inactive in female cells.Can be seen under a light microscope as a clump of heterochromatin—called a Barr bodyThus, dosage of expressed X chromosome is the same in males and females.Figure 14.18 A Barr Body in the Nucleus of a Female Cell14.4 How Is Eukaryotic Gene Transcription Regulated?Methylation of cystosines contributes to condensation and inactivation of the DNA.One gene is active: Xist (X inactivation-specific transcript). RNA that is transcribed binds to the chromosome and inactivates it—interference RNA.Figure 14.19 A Model for X Chromosome Inactivation14.4 How Is Eukaryotic Gene Transcription Regulated?An anti-Xist gene, Tsix, codes for RNA that binds to the Xist site on the active X chromosome.14.4 How Is Eukaryotic Gene Transcription Regulated?Transcription can be increased by making more copies of a gene—gene amplification.Example: The genes that code for three of the rRNAs in humans are linked and there are several hundred copies in the genome.14.4 How Is Eukaryotic Gene Transcription Regulated?Fish and frog eggs have up to a trillion ribosomes.Cells selectively amplify the rRNA gene clusters to more than a million copies.Transcribed at maximum rate, these genes produce the ribosomes for a mature egg in a few days.Figure 14.20 Transcription from Multiple Genes for rRNA14.4 How Is Eukaryotic Gene Transcription Regulated?In some cancers, a cancer-causing oncogene is amplified.The mechanism of amplification is not well understood.14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?Alternative splicing: some exons are selectively deletedDifferent proteins can be generated from the same gene.Example: The pre-mRNA for tropomyosin is spliced five different ways to produce five different forms of tropomyosin.Figure 14.21 Alternative Splicing Results in Different Mature mRNAs and Proteins14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?In humans, there are many more mRNAs than genes—mostly from alternative splicing.14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?RNA has no repair mechanisms.mRNA can be catabolyzed by ribonucleases in the cytoplasm and lysosomes.mRNAs have different stabilities—a mechanism for posttranscriptional regulation.14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?Specific AU sequences on mRNA can mark them for breakdown by a ribonuclease complex called an exosome.Signaling molecules such as growth factor are only synthesized when needed and break down rapidly. Their mRNAs have an AU sequence and are unstable.14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?Micro RNAs (about 20 bases long) bind to mRNA before it reaches a ribosome.Causes the mRNA to break down, or inhibits translation.14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?The micro RNAs start as a 70 base-pair double strand.The protein complex called dicer cuts the RNA strand.Small RNAs are under development as drugs to block gene expression of certain genes in human diseases.Figure 14.22 mRNA Inhibition by Small RNAs14.5 How Is Eukaryotic Gene Expression Regulated After Transcription?RNA editing: change in sequence after transcription and splicingInsertion of nucleotides—stretches of uracil are addedAlteration of nucleotides—an enzyme catalyzes the deamination of cytosine to from uracil.Figure 14.23 RNA Editing14.6 How Is Gene Expression Controlled During and After Translation?Translation can be modified by the G cap.If the cap is an unmodified GTP, the mRNA is not translated.Example: The stored mRNA in egg cells of tobacco hornworm moth: After the egg is fertilized, the cap is modified, and translation proceeds.14.6 How Is Gene Expression Controlled During and After Translation?Cellular conditions can control translation.Example: free iron (Fe2+) in cells is bound by ferritin When Fe2+ is low, a repressor binds to ferritin mRNA and prevents translation.As Fe2+ levels rise, Fe2+ binds to the repressor, which detaches from the mRNA.14.6 How Is Gene Expression Controlled During and After Translation?Translational control can keep a balance in the amount of subunits of proteins.Example: Hemoglobin has four globin and four heme units.If there are more heme than globin units, heme increases rate of translation of globin by removing a block to initiation of translation at ribosome. 14.6 How Is Gene Expression Controlled During and After Translation?Most proteins are modified after translation.A protein can be regulated by controlling its lifetime in the cell.In many cases, an enzyme attaches a protein called ubiquitin to a lysine in a protein targeted for breakdown.14.6 How Is Gene Expression Controlled During and After Translation?Other ubiquitin chains attach to the first one, forming a polyubiquitin complex.The whole complex then binds to a proteasome. Ubiquitin is cut off for recycling; the protein passes by three proteases that digest it.Figure 14.24 A Proteasome Breaks Down Proteins14.6 How Is Gene Expression Controlled During and After Translation?Concentrations of many proteins are determined by their degradation in proteasomes.Cyclins are degraded at the correct time in the cell cycle.Transcriptional regulators are broken down after use; to prevent gene to be always “on.” 14.6 How Is Gene Expression Controlled During and After Translation?Some viruses can take advantage of this system.Human papillomavirus (causes cervical cancer) marks protein p53 for degradation by proteasomes. p53 normally inhibits cell division.