The Little Things About the Little Things Inside of Us The Eukaryotic Genome and Its Expression

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1 The Little Things About the Little Things Inside of Us The Eukaryotic Genome and Its Expression 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. Eukaryotes have multiple chromosomes. In eukaryotes, translation and transcription are physically separated which allows many points of regulation before translation begins. Eukaryote model organisms: Yeast, Saccharomyces cerevisiae Nematode (roundworm), Caenorhabditis elegans Fruit fly, Drosophila melanogaster Thale cress, Arabidopsis thaliana The yeast (Saccharomyces cerevisiae) has 16 chromosomes; haploid content of 12 million base pairs (bp). Compartmentalization into organelles requires more genes than prokaryotes have. Some eukaryotic genes that have no homologs in prokaryotes: Genes encoding histones Genes encoding cyclin-dependent kinases that control cell division Genes encoding proteins involved in processing of mrna 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 cells The genome is eight times larger than yeasts. Drosophila melanogaster has been used extensively in genetic studies. Genome is larger than C. elegans, but has fewer genes The genome codes for more proteins than it has genes. Arabidopsis thaliana is in the mustard family. Has some genes that have homologs in C. elegans and Drosophila Also has genes that distinguish it as a plant, such as genes for photosynthesis.

2 Rice (Oryza sativa) genome has also been sequenced two subspecies Has many genes similar to Arabidopsis. Eukaryote genomes have two types of highly repetitive sequences that do not code for proteins: Minisatellites: bp, repeated several thousand times. Number of copies varies among individuals provides molecular markers. Microsatellites: 1 3 bp, copies Moderately repetitive sequences (genes): code for trna and rrna These molecules are needed in large quantities; the genome has multiple copies of the sequence. Mammals: Four different rrnas 16S, 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) 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. Four types of transposons: SINEs LINEs Retrotransposons DNA transposons SINEs (short interspersed elements) 500 bp; 15 percent of human DNA. One, Alu, is present in a million copies LINEs (long interspersed elements) 7,000 bp; about 17 percent of human DNA; some code for proteins SINEs and LINEs make an RNA copy of themselves that is a template for new DNA inserted somewhere else copy and paste mechanism. 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.

3 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. Transposons may have played a role in endosymbiosis: Genes from the once-independent prokaryotes may have moved to the nucleus by DNA transposons. 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 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. 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. 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. 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 hybridization 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.

4 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. 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. The globin gene family arose from a common ancestor gene. In humans: Alpha-globin ( -globin) three functional genes Beta-globin ( -globin) five functional genes Hemoglobin is a tetramer of two units and two units. During development, different globin genes are expressed at different times: differential gene expression. -globin is in hemoglobin of human fetus it binds oxygen more tightly than adult hemoglobin. 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 (psi) 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. 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. 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. With energy from ATP, proteins are added to form an RNA-protein complex, the spliceosome.

5 The complex cuts pre-mrna, releases introns, and splices exons together. In the disease beta thalassemia, a mutation occurs at the consensus sequence in the globin gene the pre-mrna can not be spliced correctly. Non-functional -globin mrna is produced. 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. 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. Transcriptional regulation and posttranscriptional regulation can be determined by examining mrna sequences made in different cell types. 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 trna II transcribes protein-coding genes 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). In prokaryotes, promoter has two sequences: The recognition sequence is recognized by RNA polymerase. The TATA box, where DNA begins to denature. 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. 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.

6 Regulator sequences are located upstream of the promoter. Regulator proteins bind to these sequences. Resulting complex binds to the transcription complex to activate transcription. 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. Negative regulatory sequences or silencer sequences turn off transcription by binding repressor proteins. DNA-binding proteins have four structural themes or motifs: Helix-turn-helix Zinc finger Leucine zipper Helix-loop-helix 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. 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. Transcription can also be regulated by changes in chromatin and chromosomes. 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. Histone proteins have tails with positively charged amino acids enzymes add acetyl groups: This reduces positive charges, and decreases affinity of histones for negatively charged DNA. Allows chromatin remodeling

7 Gene activation requires histone acetyl transferases to add acetyl groups. Gene repression requires histone deacetylases to remove the acetyl groups. 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. Two types of chromatin: Euchromatin contains DNA that is transcribed into mrna. Heterochromatin: genes it contains are usually not transcribed. 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. One X chromosome remains inactive in female cells. Can be seen under a light microscope as a clump of heterochromatin called a Barr body Thus, dosage of expressed X chromosome is the same in males and females. 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. An anti-xist gene, Tsix, codes for RNA that binds to the Xist site on the active X chromosome. 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. 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. In some cancers, a cancer-causing oncogene is amplified. The mechanism of amplification is not well understood. Alternative splicing: some exons are selectively deleted Different proteins can be generated from the same gene.

8 Example: The pre-mrna for tropomyosin is spliced five different ways to produce five different forms of tropomyosin. How Is Eukaryotic Gene Expression Regulated After Transcription? In humans, there are many more mrnas than genes mostly from alternative splicing. 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. 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. Micro RNAs (about 20 bases long) bind to mrna before it reaches a ribosome. Causes the mrna to break down, or inhibits translation. 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. RNA editing: change in sequence after transcription and splicing Insertion of nucleotides stretches of uracil are added Alteration of nucleotides an enzyme catalyzes the deamination of cytosine to from uracil. 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. 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.

9 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. 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. 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. 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. 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.