Study Guide Test 3
Lecture 13
DNA: transitions, transversions, insertions, deletions
Point mutations are small changes in 1 or a few base pairs
o Substitutions: one base replaces another
Transition: purine changes to purine and pyrimidine to pyrimidine
Transversion: purine to pyrimidine, and pyrimidine to purine
o Insertions and deletions: one or a few bases are inserted or deleted
Proteins: Missense, nonsense, silent, frameshift
Missense mutation: 1 base change in a protein coding region 1 amino acid change
Nonsense mutation: 1 base change stop codon, prematurely terminating the protein
Silent mutations: cause no change in protein sequence
o Alternative wildtype allele
Frameshift mutation: translation reading frame problem
o Result: inappropriate reading frame “downstream” from deletion multiple
missense substitutions
o All frameshifts eventually introduce a nonsense codon, often after one or more
missense codons
Gene expression.
Mutations in non-coding regions may affect expression
Promoter mutations: if the change prevents RNA polymerase from recognizing the
promoter, transcription will not occur = no protein = severe mutation
Some mutations in the 5’UTR may affect translation
If there is a mutation in the Shine-Dalgarno sequence the ribosome doesn’t bind no
translation no protein severe mutations
Mutations in splice junction sequences prevent splicing. Protein includes inappropriate
extra amino acids, and will include a nonsense mutation in long introns
Phenotype: loss of function, gain of function.
Loss of function: allele that produces a product with reduced function
o Complete loss of function: Null or amorphic
o Partial loss of function: hypomorphic
o Some phenotypes vary with enzyme dosage = incomplete dominance
Gain of function alleles: mutation causes extra or inappropriate activity. Almost always
dominant
o Hypermorphic: too much activity. Most often in negative cellular regulators.
Mutation results in the failure to turn off
o Neomorphic: mutation results in a new function, or function in an inappropriate
location or stage
o Antimorphic: mutant protein poisons normal proteins Topic 14 A
Different types of bacterial phenotypes.
1. Colony Morphology
a. Bacteria and unicellular eukaryotes are grown in liquid culture, or on agar (semi-
solid) plates.
b. Example: rough and smooth
2. Anabolic metabolism
a. Simple compounds + energy complex compounds
3. Catabolic metabolism
a. Compounds from environment CO2 + H2O + energy
Detecting and analyzing auxotrophic phenotypes.
Auxotrophic mutants: cannot make an essential compound (defect in an anabolic
pathway)
Auxotrophic mutants are studied by testing growth on defined media
If media contains necessary nutrients, 1 cell grows into a visible colony in 1-2 days
Identifying auxotrophic mutant
o Defined media = minimal media contains salts and an organic compound as
energy source
Detecting and analyzing nutritional mutant phenotypes.
Catabolic mutants
o Cannot break down or use particular compound as sole energy source. Defect in
catabolic (breaking down) pathways
Mutant identification:
o Test ability to grow on minimal medium with target compound as sole energy
source. Glucose is control energy source
o Most colonies are able to use lactose as sole energy source
o Lac- mutant grows when glucose is present but not when lactose is the only
energy source
Antibiotic Resistance
o Antibiotics = bacterial poisons. Kill wildtype cells. Examples:
Penicillin/amoxicillin/ampicillin
Tetracycline
Ciprofloxacin
o Antibiotic resistance:
Mutation in target protein so that antibiotic no longer binds
Enzyme with altered specificity breaks down drug
Transporter/channel proteins with altered specificity reduce intracellular
accumulation
Conditional Mutations
o Mutations in essential genes; cells that lack gene function die. Example:
Mutations in RNA polymerase, DNA polymerase, glycolysis enzymes,
ribosomal proteins o Since bacteria are haploid (1 copy of each gene), mutations must be conditional:
produce phenotype under some conditions but not others
Temperature sensitivity (most common)
Topic 14 B
Lactose metabolism and the logic of lac operon expression
Lactose is a disaccharide and is used as an energy source to:
o Import into cell
o Breakdown into monosaccharides
o Lactose catabolism only occurs when glucose is absent
LacY- and LacZ- mutants do not grow on minimal medium with lactose as sole carbon
source, i.e., cannot break down lactose. These mutants grow if glucose is present
LacY+ encodes permease, a lactose transporter (transports lactose into the cell)
LacZ+ encodes -galactosidase; breaks down lactose into glucose and galactose (each
into glycolysis)
Logic of Lac Operon
o If glucose is available, the lac genes are OFF (glucose is used in preference to
lactose)
o If glucose is not available is lactose available? If yes, lac genes are ON (lac genes
are expressed in the absence of glucose and presence of lactose)
o If glucose is not available is lactose available? If no, Lac genes OFF (enzymes for
lactose catabolism are not necessary if lactose is absent)
Structure and expression of prokaryotic operons
Operon: cluster of genes expressed as a single mRNA, and the regulatory sequences for
their expression (genes usually have similar functions)
The lac mRNA is the transcript of 3 genes, each with its own Shine-Dalgarno sequence,
AUG initiation codon and translation termination codon
Operon also contains:
o CRP-binding site
o P (promoter)
o O (operator)
CRP-binding site, promoter and operator are cis-acting sequences
The functions of trans-acting factors and cis regulatory sequences
Operator overlaps with the promoter/RNA polymerase binding site. Repressor binding
to the operator physically blocks RNA polymerase from the promoter
The negative trains-acting factor (lac repressor) is a dimer, each copy of which
recognizes the same DNA sequence
o Fits into the major groove of DNA
Positive and negative regulation
Binding of trans-acting factors is sometimes required for transcription = positive
regulation. Transcription occurs only when these are bound to cis-acting sequences
o Positive regulatory proteins = activators Binding of trans-acting factors may sometimes prevent transcription = negative
regulation. Transcription is blocked when these are bound to cis-acting sequences
o Negative regulatory proteins = repressors
Positive regulation: a bound regulatory protein is necessary for transcription
Negative regulation: a bound regulatory protein prevents transcription even in the
presence of a positive regulatory protein
Regulation of the lac operon is both positive and negative
A positive regulatory protein MUST BE bound to the CRP-binding site, and a negative
regulatory protein MUST NOT BE bound to the operator
Transcription is activated when an activator binds to regulatory DNA sequences =
positive regulation
o The activator: complex of the activator protein CRP and cyclic AMP
Binding of CRP to DNA regulatory region is controlled by cAMP availability
o CRP-cAMP complex binds to DNA and acts as an activator
o CRP without cAMP does not bind to DNA
cAMP concentration varies inversely with glucose concentration
o High [glucose] low [cAMP]
o Low [glucose] high [cAMP]
o Low glucose CRP-cAMP
o High glucose CRP only
Positive: a bound protein (CRP-cAMP) is required for transcription in the absence of
glucose
Negative: a bound protein (lac repressor) represses transcription in the absence of
lactose
o Absence of lac repressor allows transcription in the presence of lactose
Normal pattern of lac gene expression is known as “inducible”
o Off when lactose is absent
o On when lactose is present
o Occurs via negative regulation
Some mutations cause constitutive expression
o Constitutive = ON all of the time, whether lactose is absent or present
LacI+ gene encodes the lac repressor protein
o LacI: transcribed independently and at all times
LacO+: the operator = DNA regulatory sequence to which lac repressor protein binds
o When lactose is absent the repressor protein binds to the operator and prevents
transcription
o When lactose is present the repressor is inactive and does not repress
transcription
Result: transcription occurs
o Lactose inactivates the repressor by binding to its lactose-binding site which
induces a conformational change in the protein so that it no longer binds to the
operator Lactose induces by inactivating the repressor
Why do lacI and lacO mutations have constitutive phenotypes?
o The lacI+ and lacO+ functions are necessary to turn off lac operon expression
lacI-: mutant repressor protein cannot bind to operator
lacO-: mutant operator DNA sequence cannot be recognized by repressor
protein
Analysis of mutants in haploid and partial diploid cells led to an understanding of regulatory
mechanisms.
Since lacI- and lacO- have the same phenotype, how was the cis-trans model for
regulation discovered?
o [Cis-trans: repressor protein (trans) binds to operator (cis)]
Plasmids: small circular “optional” chromosomes
o Plasmid that determines mating type (F factor)
o Plasmids with antibiotic resistance genes
F factor may integrate into chromosome
Imprecise “excision” carries some chromosome genes on the F factor (now called F’
factor)
o Example: integration near the lac operon
o F’ factor contains the lac operon (F’ lac)
o Transfer of F’ lac to a normal strain by mating creates a strain with 2 copies of lac
operon = merodiploid
Topic 15
Chromatin structure and effects on gene expression
Eukaryotic nuclear DNA is packaged with protein
o Combination of DNA + protein = chromatin
Protein: histones and non-histone proteins
o Histones
5 protein types. Small (~100 amino acids), basic (high content of arginine
and lysine)
o Non-histone proteins
Thousands of types. RNA polymerase, splicing factors, regulatory
proteins
Nucleosome: fundamental unit of chromatin. Structure:
o 8 core histone proteins, 2 of H2A, H2B, H3 and H4
o ~160 base pairs of DNA wraps around the outside
th
5 histone, H1, associates with DNA entering and leaving
`40 base pairs of DNA between nucelosomes (= linker DNA)
DNA packaging:
o All chromosomes: 2 meters of DNA/cell nucleus of 6 M diameter
o Average single chromosome: 4cm DNA molecule 10 M chromosome “inactive” gene: promoter is bound by nucleosomes and inaccessible
o Activating transcription requires rearranging nucleosomes to expose promoter
Modification to histone proteins “tails” (extend from nucleosome)
o Acetylation: acetyl groups (acid) partially neutralize histones (basic); promoter
looser, open configuration
o Histone acetyl transferases (HATs) (family of acetylating enzymes)
Open configuration spreads: HATs bind to acetylated histones and
acetylate neighboring histones
o Methylation: more complex. Effect on gene expression can be positive
(activating) or negative (repressing)
Heterochromatin: dense, darkly-staining chromosome regions. Visible in mitotic
chromosomes and sometimes interphase nuclei
o Chromatin in heterochromatic regions is densely packed and not transcribed
o Consequences of gene silencing in heterochromatin
Centromeres: no consequences. Don’t contain any genes
Mammalian X: equalizes gene dose
Euchromatin: the rest of the genome, i.e., not heterochromatin
The types of cis-acting sequences
Cis-acting sequences: DNA sequences to which proteins bind to regulate transcription
Two classes:
o Promoter (AKA basal promoter): near transcription start site
Major common feature: TATA box = sequence TATATAA. 25-30 base
pairs upstream from transcription start site
o Enhancers: variable in position and distance. Can be thousands of base pairs
away from gene. Upstream or downstream, or in introns. A gene may have one
or many. Function not dependent on distance or orientation from gene
Types of transcription factors, their interactions with cis-sequences and their functions
Transcription factors: proteins that regulate transcription by binding to cis-sequences
o Basal factors: bind to the promoter
o Enhancer-binding transcription factors:
Activators (positive regulation)
Repressors (negative regulation)
Complex of basal factors binds to the promoter and recruits’ RNA polymerase
o TATA-binding protein (TBP)
o TATA-associated factors (TAFs)
o Also mediator proteins
Basal factors are generic and ubiquitous. Not responsible for turning genes on/off
How enhancers are identified.
Complexity of eukaryotic transcription regulation is largely due to activator & repressor
binding to enhancers
Enhancers can be far away from gene regulated: DNA flexibility brings
activator/repressor proteins in contact with basal factors How do activators activate?
o Recruit basal factors to the promoter. Stabilize binding of RNA polymerase
o Remodel chromatin to expose the promoter
How do repressors repress?
o Recruit corepressor proteins that interfere with the basal complex
o Recruit corepressor proteins that close chromatin structure
o Interfere with and “neutralize” activators
Compete for enhancer sites
Bind to and quench activator function
Bind to activator (sequester) to prevent DNA binding
Trans-acting factors
o Transcription factors usually have three functional domains:
DNA binding domain: responsible for DNA sequence-specific binding
Dimerization domain: allows homo- or hetero-dimers. Most transcription
factors act as dimers
Activation domain: interacts with basal transcription complex
How are enhancers identified?
o Enhancers may be far away from regulated gene, at 3’ end of gene (or within
introns)
o Using recombinant DNA methods, the spacer DNA is cut into pieces, and each
piece joined to a basal promoter and reporter gene
Reporter gene: a gene that makes a distinctive, easily identifiable product
not normally present in the organism of interest
LacZ
o -gal not present in most eukaryotic cells
o Synthetic substrate X-Gal makes detection easy
Green fluorescent protein (GFP)
o Can be detected in living cells
o Insert each hybrid DNA construct into genome of the experimental organism
o Result: some constructs show -gal expression while others do not
o Conclusions: regions that do not contain enhancers show no expression of
reporter genes. Regions that contain enhancers act in positive regulation and
drives expression in the wing
o Reporter gene expression driven by specific enhancers:
LacZ reporter gene expression in Drosophilia and mouse embryos
Combinatorial control of gene expression.
Eukaryotic gene expression is combinatorial: depends on combinations of transcription
factors
Genes are expressed differentially because different cell types have different
combinations of transcription factors
Example: gene transcribed in skin and eye cells have enhancers for each tissue
o Eye cells contain appropriate activators in S phase but no G1 o Skin cells contain appropriate activators
o Liver cells don’t contain the correct combination and don’t express
Different cells have different combinations of transcription factors because…
o Of earlier gene expression (transcription factors are proteins produced by earlier
gene expression)
o Protein modification in response to cellular conditions (phosphorylation due to a
signal transduction pathway; steroid hormone binding)
Post-transcriptional regulation
Splicing
o Regulation by splicing:
Differential RNA splicing produces different mRNAs from the same
primary transcript
Regulation of mRNA stability and translation
o Small RNAs regulate mRNA stability and translation
o 3 families of small RNAs, differing in deta
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