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The Eukaryotic Genome and Its Expression

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The Eukaryotic Genome and Its Expression The Eukaryotic Genome Repetitive Sequences in the Eukaryotic Genome The Structures of Protein-Coding Genes RNA Processing Posttranscriptional Regulation Translational and Posttranslational Regulation

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The Eukaryotic Genome Eukaryotic genomes are larger than those of prokaryotes. Eukaryotic genomes have more regulatory sequences and more regulatory proteins that bind to them. Much of eukaryotic DNA is noncoding. Eukaryotes have multiple chromosomes. In eukaryotes, transcription and translation are physically separated.

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The Eukaryotic Genome The genome of the yeast Saccharomyces cerevisiae has has been sequenced and 5,600 genes found. By means of gene annotation, around 70 percent have been assigned probable roles. Yeast has become an important model for eukaryotic cells. The proportions of the yeast genome coding for specific metabolic roles have been determined.

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The Eukaryotic Genome Both E. coli (a prokaryote) and yeast (a eukaryote) use about the same number of genes for cell survival. Yeast has many more genes for protein targeting. Eukaryotes require a greater number of genes because of the compartmentalization of the cells, confirming that eukaryote cells are structurally more complex than prokaryote cells.

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Genes encoding histones

The Eukaryotic Genome

Genes for other types of proteins that are present in eukaryotes but have no homologs in prokaryotes include:

Genes encoding cytoskeletal and motor proteins such as actin and tubulin Genes encoding cyclin-dependent kinases that control cell division Genes encoding proteins involved in the processing of RNA

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The Eukaryotic Genome Caenorhabditis elegans, a small nematode, has become a model for multicellular organisms. The genome of C. elegans has been sequenced and contains about 19,000 protein-coding genes. About 3,000 genes in the worm have homologs in yeast. These genes are the ones considered essential to all eukaryotes. Many of the remaining 16,000 genes perform roles related to multicellularity.

Table 14.3 C. elegans Genes Essential to Multicellularity

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The Eukaryotic Genome

Drosophila melanogaster is much larger than C. elegans, having 10 times more cells, but the genome has fewer protein-coding genes than C. elegans. C. elegans has more copies of related genes than Drosophila does. About half of the fly genes have mammalian homologs. The fly genome contains 177 genes whose sequences are known to be directly involved in human diseases, such as cancer. The roles of such genes are often more easily studied in the fly than in humans.

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The Eukaryotic Genome The puffer fish, Fugu rubripes, has a very compact genome consisting of about 30,000 genes. The human genome has about the same number of genes in eight times the amount of DNA. The human and puffer fish genomes have many similar genes; the puffer fish genome is an abridged version of the human genome. Repetitive DNA sequences, which make up 40 percent of the human genome, are present in much smaller proportions in the puffer fish genome.

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The Eukaryotic Genome The thale cress, Arabidopsis thaliana, has a small genome and is a model organism for study by plant biologists. The DNA sequence contains about 26,000 protein-coding genes, many of which are duplicates of other genes. Many of these genes have homologs in the fruit fly and roundworm, suggesting that plants and animals have a common ancestor. Arabidopsis also has genes unique to plants, such as those for cell walls and photosynthesis.

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The Eukaryotic Genome Rice, Oryza sativa, has many genes similar to Arabidopsis. The genomes of different subspecies of rice have been sequenced, and each has particular genes that make it unique. Analyses of these genes will lead to improvements in this and other grain crops.

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Repetitive Sequences in the Eukaryotic Genome

Three types of highly repetitive sequences are found in eukaryotes: Satellites are 5 to 50 bp long, repeated side by side up to a million times. Minisatellites are 12 to 100 bp long and repeated several thousand times. Individuals in a population can vary in the number of copies. Microsatellites are 1 to 5 bp and present in 10 to 50 copies per cluster. They are scattered all over the genome.

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Repetitive Sequences in the Eukaryotic Genome Telomeres are moderately repetitive sequences at the end of the chromosomes. They are not transcribed into RNA. However, some moderately repetitive DNA sequences code for tRNAs and rRNAs. The genome has multiple copies of these coding regions so that tRNAs and mRNAs can be produced in amounts needed by most cells.

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Repetitive Sequences in the Eukaryotic Genome In mammals there are four different rRNA molecules that make up the ribosome: 18S, 5.8S, 28S, and 5S. The 18S, 5.8S, and 28S rRNAs are transcribed as a single precursor RNA, which is twice the size of all three ultimate products. There are 280 copies of sequences coding for the transcript located in clusters on five different chromosomes.

Figure 14.2 A Moderately Repetitive Sequence Codes for rRNA

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Repetitive Sequences in the Eukaryotic Genome

Some moderately repetitive DNA sequences are transposons of which there are four main types. SINEs are short interspersed elements up to 500 bp long. They are transcribed but not translated. LINEs are long interspersed elements up to 7,000 bp long. Some are transcribed and translated into proteins. Retrotransposons, constituting about 17 percent of the human genome, also make an RNA copy when they move. DNA transposons do not use an RNA intermediate, but actually move to a new spot without replicating.

Figure 14.3 DNA Transposons and Transposition

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Repetitive Sequences in the Eukaryotic Genome Beneficial roles for transposons are unknown. They may be cellular parasites that simply replicate themselves. Insertion of a transposon into a functional gene can disable it or alter its transcription rate. Insertions in a germ cell line can result in new mutations. If insertion occurs in a somatic cell, cancer may result. Transposition increases genetic variation by shuffling genetic material and creating new genes. Transposons may have played a role in the evolution of cell organelles.

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The Structures of Protein-Coding Genes Many protein-coding genes in eukaryotes are single-copy DNA sequences. Unlike most prokaryotes, however, eukaryotes have genes with noncoding internal sequences. Eukaryotes also form gene families with structurally and functionally related cousins in the genome.

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The Structures of Protein-Coding Genes Genes have three types of noncoding sequences: The promoter occurs at the beginning of the gene and is the site where RNA polymerase begins transcription. The terminator occurs at the end of the gene and signals the end of transcription. Noncoding sequences called introns are interspersed with the coding regions, called exons.

Figure 14.4 The Structure and Transcription of a Eukaryotic Gene

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The Structures of Protein-Coding Genes The entire sequence, including introns, is transcribed. The resulting RNA is the primary transcript, or pre-mRNA. The transcripts of the introns are removed from the pre-RNA and the transcripts of the exons are spliced together, resulting in mature mRNA. Nucleic acid hybridization can be used to determine the location of introns in DNA. This method was also used in the initial discovery of introns.

Figure 14.5 Nucleic Acid Hybridization

Figure 14.6 Nucleic Acid Hybridization Revealed Noncoding DNA (Part 1)

Figure 14.6 Nucleic Acid Hybridization Revealed Noncoding DNA (Part 2)

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The Structures of Protein-Coding Genes About half of all eukaryotic protein-coding genes have a single copy in the haploid genome. The rest have multiple copies. Pseudogenes () are inexact, nonfunctional copies of genes, often found near the functional copy.

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The Structures of Protein-Coding Genes Sometimes copies of genes are functional, but slightly different. A set of duplicated or related genes is called a gene family. DNA sequences in gene families vary, but as long as one member retains the original DNA sequence, the other members can mutate without negative effects. These extra genes provide material for evolution. If the mutated gene is useful, it will be selected for in succeeding generations.

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The Structures of Protein-Coding Genes The gene family encoding the globins is an example. Humans have three -globins and five -globins. During development, different members of the globin gene family are expressed at different times and in different tissues.

Figure 14.8 Differential Expression in the Globin Gene Family

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The Structures of Protein-Coding Genes The globin gene family also includes nonfunctional pseudogenes. These black sheep family members result from mutations that cause loss of function. As long as some members of a gene family are functional and pseudogenes are not actively detrimental, there appears to be little selective pressure to eliminate the pseudogenes.

Figure 14.7 The Globin Gene Family

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RNA Processing The first two steps of processing pre-mRNA take place in the nucleus: The G cap, a modified GTP, is added to the 5 end. It facilitates the binding of mRNA to the ribosome and protects the mRNA from being digested by ribonucleases. A poly A tail is added to the 3 end. It is 100 to 300 residues of adenine (poly A) in length.

Figure 14.9 Processing the Ends of Eukaryotic Pre-mRNA

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RNA Processing RNA splicing removes the introns and splices the exons together: At the boundaries between introns and exons are consensus sequences. A small ribonucleoprotein particle (snRNP) binds to the consensus sequence at the 5 exonintron boundary. Another snRNP binds near the 3 exonintron boundary. Then other proteins bind, forming a large RNA protein complex called a spliceosome. This complex cuts the RNA, releases the introns, and joins the ends of the exons.

Figure 14.10 The Spliceosome, an RNA Splicing Machine (Part 1)

Figure 14.10 The Spliceosome, an RNA Splicing Machine (Part 2)

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Transcriptional Regulation of Gene Expression Each cell in a multicellular organism contains all the genes of the organisms genome. For normal development, the expression of genes must be regulated. Regulation of gene expression can occur at many points during development. Some mechanisms result in the selective transcription of specific genes.

Figure 14.11 Potential Points for the Regulation of Gene Expression in Eukaryotes (Part 1)

Figure 14.11 Potential Points for the Regulation of Gene Expression in Eukaryotes (Part 2)

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Transcriptional Regulation of Gene Expression With few exceptions, all cells in an organism have the same genes, but they express them differently. For example, both brain and liver cells transcribe housekeeping genes that code for enzymes and other molecules essential to the survival of all cells. However, liver cells transcribe some genes for liverspecific proteins, and brain cells transcribe some genes for brain-specific proteins. The difference in the production of proteins is due to differential transcription.

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Transcriptional Regulation of Gene Expression

Unlike prokaryotes, in which related genes are transcribed in units called operons, eukaryotes tend to have solitary genes. Eukaryotes have three different RNA polymerases: RNA polymerase II transcribes protein-coding genes to mRNA. RNA polymerase I transcribes rRNA coding sequences. RNA polymerase III transcribes tRNA and small nuclear RNAs.

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Transcriptional Regulation of Gene Expression Most eukaryotic genes have other DNA sequences that regulate transcription. In prokaryotes, a single peptide subunit can cause RNA polymerase to recognize the promoter; in eukaryotes many different proteins are involved in initiating transcription.

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Transcriptional Regulation of Gene Expression Transcription factors are regulatory proteins required for transcription in eukaryotes. RNA polymerase II does not bind until several other proteins, such as TFIID, have already bound the proteinDNA complex. Some DNA sequences, such as the TATA box, are common to most promoters; others are unique to only a few genes. Transcription factors play an important role in cell differentiation during development.

Figure 14.12 The Initiation of Transcription in Eukaryotes (Part 1)

Figure 14.12 The Initiation of Transcription in Eukaryotes (Part 2)

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Transcriptional Regulation of Gene Expression In addition to the promoter, nearby regulator sequences also affect transcription by binding regulator proteins that activate RNA polymerase. Much farther away are enhancer regions, which bind activator proteins and strongly stimulate the transcription complex. Negative regulatory regions of DNA called silencers bind proteins called repressors and turn off transcription. Thus they have the opposite effect of enhancers.

Figure 14.13 The Roles of Transcription Factors, Regulators, and Activators (Part 1)

Figure 14.13 The Roles of Transcription Factors, Regulators, and Activators (Part 2)

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Transcriptional Regulation of Gene Expression

In eukaryotes, genes on different chromosomes may require coordination. Regulation of various genes can be coordinated if all have the same regulatory sequences that bind to the same activators and regulators. One example is the stress response element in plants. Stress response elements near each of the scattered genes stimulate RNA synthesis. RNA then codes for proteins needed for water conservation.

Figure 14.14 Coordinating Gene Expression

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Transcriptional Regulation of Gene Expression Key to transcription regulation in eukaryotes is the binding of protein to specific DNA sequences. Proteins need to recognize and bind appropriate sites. There are four different structural themes or motifs for proteinDNA interactions: Helix-turn-helix Zinc finger Leucine zipper Helix-loop-helix

Figure 14.15 Protein-DNA Interactions (Part 1)

Figure 14.15 Protein-DNA Interactions (Part 2)

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Transcriptional Regulation of Gene Expression Other mechanisms that regulate transcription act on the structure of chromatin and chromosomes. The packaging of DNA by the nuclear proteins in chromatin can make DNA physically inaccessible to RNA polymerase and associated components.

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Transcriptional Regulation of Gene Expression Nucleosomes inhibit initiation and elongation of transcription. Nucleosomes are inactivated by two protein complexes in a process called chromatin remodeling. Nucleosome disaggregation occurs by acetylation of amino groups on the histones, and is associated with the activation of genes. Nucleosomes reform by deacetylation of the amino groups, and is associated with gene deactivation.

Figure 14.16 Local Remodeling of Chromatin for Transcription

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Transcriptional Regulation of Gene Expression Two different kinds of chromatin can be distinguished by staining the interphase nucleus. Euchromatin stains lightly. It contains DNA that is transcribed into mRNA. Heterochromatin stains densely and is generally not transcribed. Any genes in heterochromatin are thus inactivated.

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Transcriptional Regulation of Gene Expression Heterochromatin is in found in the inactive X chromosome of mammals. One of the X chromosomes in each cell of a female is inactivated early in development. The chromosome remains condensed and appears as a Barr body under the microscope. Condensation physically prevents DNA from being transcribed. Methylation of cytosine on DNA may be involved with the inactivation.

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Transcriptional Regulation of Gene Expression The inactive X has one gene that is only lightly methylated and transcriptionally active, called Xist. The RNA transcribed from Xist is not an mRNA and remains in the nucleus. It binds the X chromosome that transcribes it and triggers inactivation. This RNA transcript is called interference RNA (RNAi).

Figure 14.18 A Model for X Chromosome Inactivation

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Transcriptional Regulation of Gene Expression Some gene expression is regulated by DNA rearrangement. Saccharomyces cerevisiae has two mating types, a and . All cells have alleles for both types, but only one is expressed at at time. The alleles have separate locations on the chromosomes, and are separate from the MAT locus. The mating type of a given yeast cell depends on which copy, a or , exists at the MAT site. Alleles at the MAT site can be moved in and out.

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Transcriptional Regulation of Gene Expression One cell can make more proteins than another cell by making more copies of a gene, a process called gene amplification. Mature frog and fish eggs have up to a trillion ribosomes, which are used for massive protein synthesis following fertilization. To make this number, ribosomal rRNA gene clusters are selectively amplified and copied until there are a million copies in just one cell. Later, after cell division begins, the number of copies returns to normal. The mechanism for this selective amplification of a single gene is not clearly understood.

Figure 14.19 Transcription from Multiple Genes for rRNA

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Posttranscriptional Regulation There are many ways in which gene expression can be regulated after transcription. Pre-mRNA can be processed in the nucleus by cutting and splicing. The longevity of mRNA in the cytoplasm can also be regulated.

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Posttranscriptional Regulation Alternative splicing of a specific pre-mRNA can generate different proteins from a single gene. For example, cells in five different tissues splice the pre-mRNA for the structural protein tropomyosin into five different mRNAs. As a result, each of the five tissues in mammals (skeletal muscle, smooth muscle, fibroblast, liver, and brain) has a different form of tropomyosin.

Figure 14.20 Alternative Splicing Results in Different mRNAs and Proteins

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Posttranscriptional Regulation RNA has no repair mechanisms. Different mRNAs have different life spans, and the less time an mRNA spends in the cytoplasm, the less of its protein can be translated. Specific AU-rich nucleotide sequences within some mRNAs mark them for rapid breakdown by a ribonuclease complex called the exosome. Signaling molecules, such as growth factors, are made only when needed and then break down rapidly.

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Posttranscriptional Regulation RNA editing can be used to change the sequence of mRNA after transcription. This editing can take place by either the insertion of nucleotides to the mRNA sequence or the alteration of nucleotides in the mRNA.

Figure 14.21 RNA Editing

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Translational and Posttranslational Regulation Proteins can regulate translation by binding to mRNA in the cytoplasm. This is important for long-lived mRNAs. It prevents the production of unnecessary proteins. For example, cyclin, which stimulates the cell cycle, must be shut off after it has done its job. If not, inappropriate cell division may lead to a tumor.

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Translational and Posttranslational Regulation The translation of mRNA can be regulated to control levels of certain proteins. 1. Regulation by the G cap: An mRNA capped with an unmodified GTP is not translated. These mRNAs can be stored and modified later when the proteins are needed.

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Translational and Posttranslational Regulation 2. Regulation of ferritin, an iron storage protein: When excess iron is present, ferritin synthesis increases, but the amount of ferritin mRNA remains constant. When iron is low, a translational repressor protein binds to ferritin mRNA and prevents translation. When iron levels rise, excess iron binds to the repressor and alters its structure, causing it to detach from the mRNA. Translation then proceeds.

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Translational and Posttranslational Regulation 3. Regulation of hemoglobin: Hemoglobin consists of four globin units and four heme pigments. If globin synthesis does not equal heme synthesis, some heme stays free in the cell. Excess heme in the cell increases the rate of translation of globin mRNA by removing a block to initiation of translation at the ribosome.

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Translational and Posttranslational Regulation Regulating the lifetime of a protein is a way to control its actions. Proteins identified for breakdown are often linked to the protein ubiquitin. The proteinubiquitin complex then binds to a complex called a proteasome, nicknamed the molecular chamber of doom. The protein is cleaved from the ubiquitin and three different proteases digest it. Overall, concentrations of proteins depend on rates of synthesis and rates of digestion.

Figure 14.22 The Proteasome Breaks Down Proteins