Download 1) Regulation of Gene expression 2) Genomes 3

9.0 Regulation and Recombinant DNA
Related Sadava’s chapters:
1)  Regulation of Gene expression
2)  Genomes
3)  Recombinant DNA
Additional source: “A genetic switch”
Mark Ptashne
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
The rate of a metabolic pathway can be
regulated in two ways:
• Allosteric regulation of enzyme-catalyzed
reactions allows rapid fine-tuning.
• Regulation of protein synthesis
(regulation of the concentration of
enzymes) is slower but conserves
resources.
9.1 Regulation of Gene expression
In case of lack of precursor whose metabolism involves a
particular protein, prokaryotes can:
•  Inhibit the protein’s function
•  Hydrolyze the protein after it is made
•  Prevent mRNA translation at the ribosome
•  Hydrolyze mRNA, preventing translation
•  Down-regulate mRNA transcription
Prokaryotes generally use the most efficient way—down
regulating mRNA transcription.
Less energy is wasted as it is early in protein synthesis.
9.1 Regulation of Gene expression
E. coli must adapt quickly to food supply changes and
can use glucose or lactose as a sole source of energy.
Uptake and metabolism of lactose involve three proteins:
•  β-galactoside permease—a carrier protein that moves
sugar into the cell
•  β-galactosidase—an enzyme that hydrolyses lactose
•  β-galactoside transacetylase—transfers acetyl groups
to certain β-galactosides
If E.coli is grown with glucose but no lactose present, no
enzymes for lactose conversion are produced.
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
Besides the promoter, other sequences
bind regulatory proteins that interact with
RNA polymerase and regulate rate of
transcription.
Some bind positive regulators—
enhancers; others repressors—
silencers.
The combination of factors present
determines the rate of transcription.
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
Three criteria for DNA recognition by a
protein motif:
• Fits into major or minor groove
• Has amino acids that can project into
interior of double helix
• Has amino acids that can bond with
interior bases
9.1 Regulation of Gene expression
• Genes to be regulated simultaneously
may be far apart or on different
chromosomes.
• Gene expression is coordinated if they
have the same regulatory sequences
that bind same transcription factors.
• Example: A regulatory sequence in plant
genes called stress response element
(SRE)—encodes for proteins needed to
cope with drought.
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
• Epigenetics refers to changes in
expression in a gene or set of genes,
without a change in the DNA sequence.
• Changes are sometimes heritable and
stable, but are reversible.
• Includes two processes: DNA
methylation and chromosomal protein
alterations.
9.1 Regulation of Gene expression
• Some cytosine residues in DNA are
modified by adding a methyl group
covalently to the 5′ carbon—forms 5′ methylcytosine.
• DNA methyltransferase catalyzes the
reaction—usually in adjacent C and G
residues.
• Regions are called CpG islands—often
in promoters.
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
Effects of DNA methylation:
• Methylated DNA attracts proteins that
are involved in repression of
transcription and can inactivate DNA
• Important in development—early
demethylation allows many genes to
become active
• In cancer, misregulation can occur in
oncogenes and tumor suppressors
9.1 Regulation of Gene expression
Chromatin remodeling is the alteration of
chromatin structure.
•  Nucleosomes contain DNA and histones in a tight
complex, inaccessible to RNA polymerase.
•  Each histone has a positively charged “tail” at its N
terminus with amino acids.
•  Histone acetyltransferases change the tail’s charge
by adding acetyl groups to the amino acids.
•  This opens up nucleosomes and activates
transcription.
9.1 Regulation of Gene expression
9.1 Regulation of Gene expression
The “histone code”—histone
modifications affect gene activation and
repression:
• Histone acetylation activates
transcription
• Histone methylation affects gene
expression depending on which amino
acid is involved (several lysines in
particular)
9.2 Genomes
9.2 Genomes
Genome sequence information is used to identify:
•  Open reading frames, or coding regions
•  Amino acid sequences of proteins
•  Regulatory sequences
•  RNA genes
• Other noncoding sequences
In comparative genomics, newly sequenced
genomes are compared with sequences from other
organisms. This can give information about the
functions of sequences, and is used to trace
evolutionary relationships.
9.2 Genomes
Functional genomics assigns
functions to the products of genes.
H. influenzae chromosome has 1,738
open reading frames. When it was
first sequenced, only 58 percent
coded for proteins with known
functions.
Genome sequencing provides
insights into microorganisms that
cause human diseases and reveal
new methods to combat them.
Sequencing also reveals
relationships between pathogenic
organisms, suggesting that genes
may be transferred between
different strains.
9.2 Genomes
DNA can also be analyzed
directly from environmental
samples.
Metagenomics—genetic
diversity is explored without
isolating intact organisms.
Shotgun sequencing is used
to detect presence of known
microbes, as well as
heretofore unidentified
organisms.
It is estimated that 90 percent
of the microbial world has
been invisible to biologists
and is only now being
revealed by metagenomics.
9.2 Genomes
Mycoplasma genitalium can survive in
the laboratory with only 382
functional genes.
In 2010 a synthetic bacterial genome
was synthesized and transplanted
into a M. capricolum cell
This could have many useful benefits,
but also raises many ethical
concerns.
9.2 Genomes
9.2 Genomes
9.2 Genomes
There are major differences between eukaryotic and
prokaryotic genomes:
•  Eukaryotic genomes are larger and have more
protein-coding genes
•  Eukaryotic genomes have more regulatory
sequences. Greater complexity requires more
regulation
•  Much of eukaryotic DNA is noncoding, including
introns, gene control sequences, and repeated
sequences
•  Eukaryotes have multiple chromosomes; each must
have an origin of replication (ori), a centromere, and a
telomeric sequence at each end
9.2 Genomes
9.2 Genomes
9.2 Genomes
Eukaryotes have closely related genes
called gene families.
These arose over evolutionary time
when different copies of genes
underwent separate mutations.
For example: Genes encoding the
globin proteins all arose from a single
common ancestral gene.
9.2 Genomes
9.2 Genomes
9.2 Genomes
• Moderately repetitive sequences
are repeated 10–1,000 times
• They include the genes for tRNAs
and rRNAs
• Single copies of the tRNA and
rRNAgenes would be inadequate to
supply the large amounts of these
molecules needed by cells
9.2 Genomes
Some interesting facts about the human genome:
•  Protein-coding regions make up less than 2%, about
24,000 genes
•  Each gene codes for several proteins due to
posttranscriptional mechanisms (e.g., alternative splicing)
•  An average gene has 27,000 base pairs
•  Over 50 percent of the genome is transposons and other
repetitive sequences
•  97 percent of the genome is the same in all people
•  The chimpanzee shares 95% of the human genome and
the rhesus macaque 91%.
9.2 Genomes
9.2 Genomes
Genetic variation can affect an individual’s response
to a particular drug.
Pharmacogenomics makes it possible to predict
whether a drug will be effective. The aim is to
personalize drug treatments.
9.3 Recombinant DNA
Restriction endonucleases are
bacterial enzymes used to cut DNA.
DNA ligase catalyzes the joining of
DNA fragments.
Restriction enzymes and DNA ligase
are used to cut DNA into fragments
and then splice them together in new
combinations.
9.3 Recombinant DNA
9.3 Recombinant DNA
9.3 Recombinant DNA
9.3 Recombinant DNA
9.3 Recombinant DNA
9.3 Recombinant DNA
Biotechnology is the use of living
cells or organisms to produce
materials useful to people.
Examples:
• Using yeasts to brew beer and wine
• Using bacteria to make cheese,
yogurt, etc.
• Using microbes to produce
antibiotics, alcohol, and other
products
9.3 Recombinant DNA
After wounds heal, blood clots are
dissolved by plasmin. Plasmin is
stored as an inactive form called
plasminogen.
Conversion of plasminogen is activated
by TPA.
TPA can be used to treat strokes and
heart attacks, but large quantities are
needed. Can be made using
recombinant DNA technology.
9.3 Recombinant DNA
Pharming: Production of pharmaceuticals in farm
animals or plants.
Example: Transgenes are inserted next to the
promoter for lactoglobulin (a protein in milk). The
transgenic animal then produces large quantities
of the protein in its milk.
Human growth hormone (for children suffering
deficiencies) can now be produced by transgenic
cows.
Only 15 such cows are needed to supply all the
children in the world suffering from this type of
dwarfism.
9.3 Recombinant DNA
Crops with improved nutritional characteristics:
•  Rice does not have β-carotene, but does have a precursor
molecule
•  Genes for enzymes that synthesize β-carotene from the
precursor are taken from daffodils and inserted into rice by the Ti
plasmid
9.3 Recombinant DNA
Recombinant DNA is also used to adapt a crop plant to an
environment.
Example: Plants that are salt-tolerant.
Genes from a protein that moves sodium ions into the central
vacuole were isolated from Arabidopsis thaliana and inserted
into tomato plants.
9.3 Recombinant DNA
Concerns over biotechnology:
•  Genetic manipulation is an unnatural interference
in nature
•  Genetically altered foods are unsafe to eat
•  Genetically altered crop plants are dangerous to
the environment
•  The complexity of the biological world makes it
impossible to predict all potential environmental
effects of transgenic organisms.
•  In fact, some spreading of transgenes has been
detected.