The Eukaryotic Genome and Its Expression

1
The Eukaryotic Genome and Its Expression
2
14 The Eukaryotic Genome and Its Expression

• 14.1 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?

3
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.

4
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.

5
Figure 14.1 Eukaryotic mRNA is Transcribed in the
Nucleus but Translated in the Cytoplasm
6
Table 14.1
7
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Eukaryote model organisms
• Yeast, Saccharomyces cerevisiae
• Nematode (roundworm), Caenorhabditis elegans
• Fruit fly, Drosophila melanogaster
• Thale cress, Arabidopsis thaliana

8
14.1 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.

9
Table 14.2
10
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• 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

11
14.1 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 cells
• The genome is eight times larger than yeasts.

12
Table 14.3
13
14.1 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
  genes
• The genome codes for more proteins than it has
  genes.

14
Figure 14.2 Functions of the Eukaryotic Genome
15
14.1 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 Drosophila
• Also has genes that distinguish it as a plant,
  such as genes for photosynthesis.

16
Table 14.4
17
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Rice (Oryza sativa) genome has also been
  sequencedtwo subspecies
• Has many genes similar to Arabidopsis.

18
Table 14.5
19
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Eukaryote genomes have two types of highly
  repetitive sequences that do not code for
  proteins
• Minisatellites 1040 bp, repeated several
  thousand times. Number of copies varies among
  individualsprovides molecular markers.
• Microsatellites 13 bp, 15100 copies

20
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Moderately repetitive sequences (genes) code for
  tRNA and rRNA
• These molecules are needed in large quantities
  the genome has multiple copies of the sequence.

21
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• 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)

22
Figure 14.3 A Moderately Repetitive Sequence
Codes for rRNA
23
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Other moderately repetitive sequences can move
  from place to place in the genometransposons.
• Transposons make up 40 percent of human genome,
  only 310 percent in other sequenced eukaryotes.

24
14.1 What Are the Characteristics of the
Eukaryotic Genome?

• Four types of transposons
• SINEs
• LINEs
• Retrotransposons
• DNA transposons

25
14.1 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 copies
• LINEs (long interspersed elements)7,000 bp
  about 17 percent of human DNA some code for
  proteins

26
14.1 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
  elsecopy and paste mechanism.

27
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.

28
Figure 14.4 DNA Transposons and Transposition
29
14.1 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 its in a somatic cell,
  cancer can result.
• Transposons can carry genes to new
  locationsadding to genetic variation.

30
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.

31
14.2 What Are the Characteristics of Eukaryotic
Genes?

• Gene characteristics not found in prokaryotes
• Eukaryote genes contain noncoding internal
  sequences.
• Form gene familiesgroups of structurally and
  functionally related genes

32
14.2 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.

33
Figure 14.5 Transcription of a Eukaryotic Gene
(Part 1)
34
Figure 14.5 Transcription of a Eukaryotic Gene
(Part 2)
35
14.2 What Are the Characteristics of Eukaryotic
Genes?

• Protein-coding genes have noncoding
  sequencesintrons.
• The coding sequences are extrons.
• Transcripts of introns appear in the pre-mRNA,
  they are removed from the final mRNA.

36
14.2 What Are the Characteristics of Eukaryotic
Genes?

• Nucleic acid hybridization reveals introns.
• Target DNA is denatured then incubated with a
  probea nucleic acid strand from another source.
• If the probe has a complementary sequence, base
  pairing forms a hybrid.

37
Figure 14.6 Nucleic Acid Hybridization
38
14.2 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 occurthe introns.
• If pre-mRNA was used, resulted in complete
  hybridization

39
Figure 14.7 Nucleic Acid Hybridization Revealed
the Existence of Introns (Part 1)
40
Figure 14.7 Nucleic Acid Hybridization Revealed
the Existence of Introns (Part 2)
41
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.

42
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.

43
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.

44
14.2 What Are the Characteristics of Eukaryotic
Genes?

• The globin gene family arose from a common
  ancestor gene.
• In humans
• Alpha-globin (a-globin)three functional genes
• Beta-globin (ß-globin)five functional genes
• Hemoglobin is a tetramer of two a units and two
  ß units.

45
Figure 14.8 The Globin Gene Family
46
Figure 3.9 Quaternary Structure of a Protein
47
14.2 What Are the Characteristics of Eukaryotic
Genes?

• During development, different globin genes are
  expressed at different times differential gene
  expression.
• ?-globin is in hemoglobin of human fetusit binds
  oxygen more tightly than adult hemoglobin.

48
Figure 14.9 Differential Expression in the Globin
Gene Family
49
14.2 What Are the Characteristics of Eukaryotic
Genes?

• Some gene families have pseudogenesresult from a
  mutation that results in loss of function.
• Pseudogenes may lack a promoter, or recognition
  sites for removal of introns.
• Designated by ? (psi)

50
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.

51
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 adeninesthe tail.
• May assist in export from nucleus important for
  stability of mRNA.

52
Figure 14.10 Processing the Ends of Eukaryotic
Pre-mRNA (Part 1)
53
Figure 14.10 Processing the Ends of Eukaryotic
Pre-mRNA (Part 2)
54
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.

55
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.

56
Figure 14.11 The Spliceosome An RNA Splicing
Machine
57
14.3 How Are Eukaryotic Gene Transcripts
Processed?

• In the disease beta thalassemia, a mutation
  occurs at the consensus sequence in the ß-globin
  genethe pre-mRNA can not be spliced correctly.
• Non-functional ß-globin mRNA is produced.

58
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.

59
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.

60
Figure 14.12 Potential Points for the Regulation
of Gene Expression (Part 1)
61
Figure 14.12 Potential Points for the Regulation
of Gene Expression (Part 2)
62
Figure 14.12 Potential Points for the Regulation
of Gene Expression (Part 3)
63
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.

64
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 tRNA
• II transcribes protein-coding genes

65
14.4 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).

66
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.

67
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.

68
Figure 14.13 The Initiation of Transcription in
Eukaryotes (Part 1)
69
Figure 14.13 The Initiation of Transcription in
Eukaryotes (Part 2)
70
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.

71
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.

72
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Enhancer sequences are farther awayup to 20,000
  bp.
• Activator proteins bind to enhancer sequences,
  which stimulates transcription complex. Mechanism
  not known perhaps by DNA bending.

73
Figure 14.14 Transcription Factors, Regulators,
and Activators (Part 1)
74
Figure 14.14 Transcription Factors, Regulators,
and Activators (Part 2)
75
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Negative regulatory sequences or silencer
  sequences turn off transcription by binding
  repressor proteins.

76
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• DNA-binding proteins have four structural themes
  or motifs
• Helix-turn-helix
• Zinc finger
• Leucine zipper
• Helix-loop-helix

77
Figure 14.15 ProteinDNA Interactions (Part 1)
78
Figure 14.15 ProteinDNA Interactions (Part 2)
79
Figure 14.15 ProteinDNA Interactions (Part 3)
80
Figure 14.15 ProteinDNA Interactions (Part 4)
81
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
  configurationbinding of repressor prevents other
  proteins from binding and initiating
  transcription.

82
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Regulation of genes that are far apart or on
  different chromosomesgenes 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.

83
Figure 14.16 Coordinating Gene Expression (Part 1)
84
Figure 14.16 Coordinating Gene Expression (Part 2)
85
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Transcription can also be regulated by changes in
  chromatin and chromosomes.

86
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.

87
Figure 14.17 Local Remodeling of Chromatin for
Transcription (Part 1)
88
Figure 14.17 Local Remodeling of Chromatin for
Transcription (Part 2)
89
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Histone proteins have tails with positively
  charged amino acidsenzymes add acetyl groups

90
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• This reduces positive charges, and decreases
  affinity of histones for negatively charged DNA.
• Allows chromatin remodeling

91
14.4 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.

92
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• The histone codehistone 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.

93
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.

94
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.

95
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 heterochromatincalled a Barr body
• Thus, dosage of expressed X chromosome is the
  same in males and females.

96
Figure 14.18 A Barr Body in the Nucleus of a
Female Cell
97
14.4 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 itinterference RNA.

98
Figure 14.19 A Model for X Chromosome Inactivation
99
14.4 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.

100
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• Transcription can be increased by making more
  copies of a genegene amplification.
• Example The genes that code for three of the
  rRNAs in humans are linked and there are several
  hundred copies in the genome.

101
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.

102
Figure 14.20 Transcription from Multiple Genes
for rRNA
103
14.4 How Is Eukaryotic Gene Transcription
Regulated?

• In some cancers, a cancer-causing oncogene is
  amplified.
• The mechanism of amplification is not well
  understood.

104
14.5 How Is Eukaryotic Gene Expression Regulated
After Transcription?

• Alternative splicing some exons are selectively
  deleted
• Different 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.

105
Figure 14.21 Alternative Splicing Results in
Different Mature mRNAs and Proteins
106
14.5 How Is Eukaryotic Gene Expression Regulated
After Transcription?

• In humans, there are many more mRNAs than
  genesmostly from alternative splicing.

107
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 stabilitiesa mechanism for
  posttranscriptional regulation.

108
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.

109
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.

110
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.

111
Figure 14.22 mRNA Inhibition by Small RNAs
112
14.5 How Is Eukaryotic Gene Expression Regulated
After Transcription?

• RNA editing change in sequence after
  transcription and splicing
• Insertion of nucleotidesstretches of uracil are
  added
• Alteration of nucleotidesan enzyme catalyzes the
  deamination of cytosine to from uracil.

113
Figure 14.23 RNA Editing
114
14.6 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.

115
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.

116
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.

117
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.

118
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.

119
Figure 14.24 A Proteasome Breaks Down Proteins
120
14.6 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.

121
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.