Eukaryotic Gene Regulation


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Eukaryotic Gene Regulation

  1. 1. Eukaryotic Gene Regulation
  2. 2. Eukaryotic vs. Prokaryotic Cells <ul><li>Eukaryotic cells have a much larger genome </li></ul><ul><li>Eukaryotes have much greater cell specialization </li></ul><ul><li>Thus eukaryotic cells contain an enormous amount of DNA that does not program the synthesis of RNA or protein </li></ul><ul><li>This requires complex organization </li></ul>
  3. 3. Eukaryotic Chromatin <ul><li>Eukaryotic chromatin is more complex than prokaryotic </li></ul><ul><li>Organization requires more packing </li></ul><ul><ul><li>DNA associates with proteins to form chromatin </li></ul></ul><ul><ul><li>Chromatin is ordered into higher structural levels </li></ul></ul>
  4. 4. Chromatin Organization <ul><li>Histones create the first level of DNA packing </li></ul><ul><li>Unfolded DNA looks like beads on a string </li></ul><ul><ul><li>Each “bead” and its DNA = nucleosome </li></ul></ul><ul><li>The beaded string undergoes higher-order packing </li></ul><ul><li>Mitotic chromosomes show several stages of chromatin coiling </li></ul><ul><li>Packaging steps are specific, so that the same genes always end up at the same place </li></ul><ul><li>Even during interphase, certain portions of chromosomes are highly condensed, an thus visible </li></ul><ul><ul><li>This is called heterochromatin to distinguish from euchromatin, “true chromatin” </li></ul></ul>
  5. 6. Non-coding DNA <ul><li>In humans, ~97% of the DNA does not code for protein or RNA </li></ul><ul><li>Some is regulatory </li></ul><ul><li>For most, the function is unknown </li></ul><ul><ul><li>Some of this is introns, some are repetitive sequences </li></ul></ul><ul><li>Repetitive Sequences </li></ul><ul><ul><li>Tandem repetitive DNA </li></ul></ul><ul><ul><ul><li>Short sequences repeated over and over </li></ul></ul></ul><ul><ul><ul><li>Some are coding and can be associated with genetic diseases </li></ul></ul></ul><ul><ul><li>Interspersed Repetitive DNA </li></ul></ul><ul><ul><ul><li>Repeat units are scattered throughout the genome </li></ul></ul></ul><ul><ul><ul><li>One group are the Alu elements </li></ul></ul></ul><ul><ul><ul><li>These are transcribed, but function is unknown </li></ul></ul></ul>
  6. 8. Multi-gene Families <ul><li>A collection of identical or very similar genes </li></ul><ul><li>Some genes are present in more than one copy, or two copies closely resemble each other </li></ul><ul><li>Likely evolved from a single ancestral gene </li></ul><ul><li>Like repetitive DNA </li></ul><ul><li>May be clustered or dispersed in the genome </li></ul><ul><li>Probably arose by repeated gene duplication, resulting from errors during DNA replication and recombination </li></ul>
  7. 9. A Family of Identical Genes for rRNA
  8. 10. The Hemoglobin Example <ul><li>Two related families of genes encode globins, the alpha and beta subunits of hemoglobin </li></ul><ul><li>Similarities suggest that the alpha and beta globins evolved from a common ancestral globin </li></ul><ul><ul><li>One family on chromosome 16 encodes for varied versions of alpha globin </li></ul></ul><ul><ul><li>The other, on chromosome 11 encodes versions of beta globin </li></ul></ul><ul><li>Different versions of each globin are expressed at different time in development, improving the flexibility of hemoglobin </li></ul>
  9. 11. Globin Gene Evolution
  10. 12. Alterations of Gene Expression <ul><li>DNA of somatic cells can be altered </li></ul><ul><li>Changes do not effect gametes so they are not passed to offspring </li></ul><ul><li>Do effect gene expression within a cell </li></ul><ul><li>These include: </li></ul><ul><ul><li>Gene amplification </li></ul></ul><ul><ul><li>Gene loss </li></ul></ul><ul><ul><li>Rearrangement </li></ul></ul>
  11. 13. Gene Amplification <ul><li>The number of copies of a gene may increase in some cells at a particular time in development </li></ul><ul><li>Example: </li></ul><ul><li>Gene for ribosomal RNA in amphibians </li></ul><ul><ul><li>The developing ovum synthesizes over a million additional copies of the rRNA gene </li></ul></ul><ul><ul><li>Enables the developing egg cell to make many more ribosomes </li></ul></ul><ul><ul><li>These are needed for the burst of protein synthesis following fertilization </li></ul></ul>
  12. 14. Gene Loss & Rearrangement <ul><li>Gene Loss </li></ul><ul><ul><li>In some insects, genes can also be selectively lost in certain tissues during development </li></ul></ul><ul><li>Genetic Rearrangement </li></ul><ul><ul><li>Shuffling of substantial stretches of DNA </li></ul></ul><ul><ul><li>Not the rearrangement of meiosis, but rearrangement in somatic cells </li></ul></ul><ul><ul><li>Rearrangement can alter gene expression </li></ul></ul>
  13. 15. Transposons <ul><li>Stretches of DNA that can move from one location to another within the genome </li></ul><ul><li>If a transposon “jumps” into the middle of a coding sequence of another gene, it can prevent normal functioning. </li></ul><ul><li>If a transposon inserts in a sequence regulating transcription, it may increase or decrease protein production </li></ul><ul><li>The transposon may also carry a gene that becomes activated if it is inserted downstream of a promoter. </li></ul>
  14. 16. Retrotransposons <ul><li>Transposable elements that move within a genome that move with a genome by means of an RNA intermediate </li></ul><ul><ul><li>A transcript of the retrotransposon DNA </li></ul></ul><ul><li>This is accomplished by reverse transcriptase </li></ul><ul><li>The Alu elements are retrotransposons </li></ul>
  15. 17. Retrotransposon Movement
  16. 18. Control of Gene Expression <ul><li>Each cell of a multi-cellular eukaryote expresses only a small fraction of its genes </li></ul><ul><li>Gene expression must be controlled to reflect differentiation (specialization) </li></ul><ul><ul><li>A typical human cell expresses only 3 – 5% of its genes at a given time </li></ul></ul><ul><li>Cells must continuously turn certain genes on and off in response to environmental signals </li></ul>
  17. 19. Steps in Gene Regulation <ul><li>Control can occur at any step in the pathway from gene to protein </li></ul><ul><li>Key stages in the expression of a protein coding gene that can be regulated: </li></ul><ul><ul><li>Unpacking DNA </li></ul></ul><ul><ul><li>Transcription </li></ul></ul><ul><ul><li>RNA processing </li></ul></ul><ul><ul><li>Translation </li></ul></ul><ul><li>Each stage is a possible control point where gene expression can be turned on or off, speeded up or slowed down </li></ul>
  18. 21. Chromatin Modification <ul><li>The physical state (packing) of DNA in or near a gene helps control whether the gene is available for transcription </li></ul><ul><li>Example: </li></ul><ul><ul><li>Genes in heterochromatin, which is highly condensed, are usually not expressed </li></ul></ul><ul><li>A gene’s location relative to nucleosomes can affect whether it is transcribed </li></ul><ul><li>Chemical modifications of chromatin help regulate transcription: </li></ul><ul><ul><li>DNA methylation </li></ul></ul><ul><ul><li>Histone acrtyylation </li></ul></ul>
  19. 22. DNA Methylation <ul><li>The attachment of methyl groups (-CH 3 ) to DNA bases after synthesis </li></ul><ul><li>Inactive DNA is generally highly methylated compared to DNA that is actively transcribed </li></ul><ul><ul><li>The same genes in different tissues are more heavily methylated in cells where they are not expressed </li></ul></ul><ul><ul><li>Demethylating inactive genes can turn them on </li></ul></ul><ul><li>May determine long term inactivation of genes </li></ul><ul><li>Methylation patterns are passed on, preserving a record of embryonic development </li></ul><ul><ul><li>May account for genomic imprinting in mammals </li></ul></ul><ul><ul><li>Methylation permanantly turns off either the maternal or paternal allele of some genes </li></ul></ul>
  20. 23. Histone Acetylation <ul><li>The attachment of acetyl groups (-COOH 3 ) to certain amino acids of histone proteins </li></ul><ul><li>May play a direct role in the regulation of gene transcription </li></ul><ul><li>When histones of a nucleosome are acetylated, they change shape so they grip the DNA less tightly </li></ul><ul><ul><li>This allows transcription enzymes easier access to the genes </li></ul></ul><ul><li>Enzymes that acetylate or deacetylate histones may be closely associated with or part of transcription factors that bind promoters </li></ul><ul><ul><li>Thus histone acetylation and gene transcription are structurally and functionally coupled </li></ul></ul>
  21. 24. Transcription Initiation <ul><li>Finer tuning of gene regulation begins with the interaction of transcription factors with DNA sequences that control specific genes </li></ul><ul><li>Once a gene is “unpacked” initiation is the most common point of control of gene expression </li></ul>
  22. 25. Eukaryotic Gene Organization <ul><li>One major difference between prokaryotic and eukaryotic genes is the presence of introns </li></ul><ul><li>A cluster of proteins called transcription initiation complex assembles on the promoter sequence at the upstream end of the gene </li></ul><ul><ul><li>RNA polymerase proceeds to transcribe the gene </li></ul></ul><ul><ul><li>The introns are removed from the primary transcript during RNA processing </li></ul></ul><ul><li>Processing pre-RNA also includes addition of a modified GTP cap to the 5’ end and a polyA tail to the 3’ end </li></ul><ul><li>Eukaryotic genes also have a relatively large number of associated control elements </li></ul><ul><ul><li>Segments of non-coding DNA that help regulate transcription by binding transcription factors </li></ul></ul>
  23. 27. The Role of Transcription Factors <ul><li>Eukaryotic RNA polymerase alone cannot initiate transcription of a gene </li></ul><ul><li>It requires transcription factors </li></ul><ul><li>Only one of the transcription factors recognizes a DNA sequence (the TATA box) within the promoter </li></ul><ul><li>Other transcription factors primarily recognize proteins and RNA polymerase </li></ul><ul><li>Only when a complete initiation complex is assembled can the polymerase move along the DNA template strand </li></ul>
  24. 28. The Role of Control Elements <ul><li>Some control elements on the DNA are close to the promoter ( proximal ) </li></ul><ul><li>More distant ( distal ) control elements are called enhancers </li></ul><ul><ul><li>May be thousands of nucleotides away or within an intron </li></ul></ul><ul><li>Bending of the DNA enables transcription factors bound to enhancers to communicate with transcription initiation complex proteins at the promoter </li></ul><ul><li>A transcription factor that binds an enhancer and stimulates transcription of a gene is called an activator </li></ul>
  25. 29. Enhancer Action
  26. 30. DNA-binding Domains <ul><li>A transcription factor generally has a DNA-binding domain </li></ul><ul><ul><li>A part of its 3D structure that binds to DNA </li></ul></ul><ul><li>There are only a few types of these domains </li></ul><ul><li>Each transcription factor has a protein-binding domain that recognizes another transcription factor </li></ul>
  27. 31. Types of DNA Binding Domains
  28. 32. Coordinately Controlled Genes <ul><li>Some genes with related functions need to be turned on or off at the same time </li></ul><ul><li>In prokaryotes such genes are often clustered into an operon </li></ul><ul><ul><li>Share a promoter and other control elements located upstream </li></ul></ul><ul><ul><li>Genes are transcribed and translated together </li></ul></ul><ul><ul><li>Not found in Eukaryotes </li></ul></ul><ul><li>Coordinate gene expression in Eukaryotes depends on association of control elements with all of the genes in a dispersed group </li></ul><ul><ul><li>Transcription factors that recognize these control elements bind to them, promoting simultaneous transcription of the genes. </li></ul></ul>
  29. 33. Post-transcriptional Mechanisms <ul><li>The expression of a protein-coding gene is measured by the amount of functional protein a cell makes </li></ul><ul><li>Gene expression may be blocked or stimulated by post-transcriptional steps </li></ul><ul><li>Post-transcriptional regulatory mechanisms can fine tune gene expression and respond to changes in the environment </li></ul><ul><li>These include: </li></ul><ul><ul><li>Alternative RNA splicing </li></ul></ul><ul><ul><li>Regulation of mRNA degradation </li></ul></ul><ul><ul><li>Control of translation </li></ul></ul><ul><ul><li>Protein processing & degradation </li></ul></ul>
  30. 34. Alternative RNA Splicing <ul><li>Different mRNA molecules are produced from the same primary transcript </li></ul><ul><li>Different RNA segments are treated as exons or introns </li></ul><ul><li>Regulatory proteins control intron-exon choices by binding to regulatory sequences within the primary transcript </li></ul>
  31. 35. Alternative RNA Splicing
  32. 36. Regulation of mRNA Degradation <ul><li>mRNA molecules in eukaryotes are longer-lived than those in prokaryotes </li></ul><ul><li>A common pathway of mRNA breakdown is enzymatic shortening of the Poly-A tail </li></ul><ul><li>This triggers enzymes that remove the 5’ cap </li></ul><ul><li>Once the cap is removed nuclease enzymes chew up the mRNA </li></ul>
  33. 37. Control of Translation <ul><li>Translation of specific mRNAs can be blocked by regulatory proteins that bind to sequences within the leader region at the 5’ end of the mRNA </li></ul><ul><ul><li>Prevents attachment of ribosomes </li></ul></ul><ul><li>Important in embryonic development </li></ul><ul><ul><li>Ovum provides a variety of mRNAs that can be translated at specific stages in development </li></ul></ul>
  34. 38. Protein Processing & Degradation <ul><li>The final opportunities for regulation are after translation </li></ul><ul><li>Eukaryotic polypeptides must often be processed to create functional proteins </li></ul><ul><ul><li>They may be cleaved, acquire sugars or phosphate groups </li></ul></ul><ul><li>Polypeptides often must be transported to specific destinations in the cell to function </li></ul><ul><li>The cell also degrades defective or damaged proteins </li></ul><ul><ul><li>To mark a protein for destruction, the cell attaches the small protein, ubiquitin </li></ul></ul><ul><ul><li>Protein complexes, called proteasomes, recognize ubiquitin and degrade the tagged protein </li></ul></ul>
  35. 39. Protein Degradation by a Proteasome