GN 311 EXAM 4
What two products result from unequal crossing over?
1. A chromosome with an insertion 2. A chromosome with a deletion
Explain how proofreading repairs replication errors.
1. An improper base is added during replication. DNA polymerase stalls replication. 2. An exonuclease from the polymerase removes the incorrect nucleotide. 3. DNA polymerase inserts the correct nucleotide and continues forward.
List the four levels of regulation in eukaryotes.
1. Changes in chromatin 2. Initiation of transcription 3. RNA processing and stability 4. Protein modification
Describe how deamination at a single site may affect DNA after replication.
1. Deamination results in a CG to TA transition in the DNA sequence (i.e., a cytosine is changed to a uracil). 2. The DNA strand with the normal sequence is replicated to produce a normal DNA molecule. The DNA strand with the uracil is replicated such that the newly-synthesized strand now has adenine adjacent to the uracil instead of guanine.
Describe how depurination at a single site may affect DNA after replication.
1. Depurination removes a base from the DNA sequence, producing an apurinic site. 2. The DNA strand that doesn't have an AP site is replicated to produce a normal DNA molecule. The newly synthesized strand of the DNA strand with the AP site has an incorrect base (A) adjacent to the AP site.
Explain how nucleotide excision repair removes lesions.
1. Enzyme complex recognizes a distortion in the helix. 2. The DNA strands are separated and held apart by single-stranded binding proteins. 3. The enzyme cleaves the sugar-phosphate bonds on both sides of the lesion/distortion. Several nucleotides are removed. 4. DNA polymerase fills in the gap and DNA ligase seals the nicks.
How can base analogs cause transitions after three rounds of replication? Use 5-bromouracil as an example.
1. First Replication: 5-bromouracil may become incorporated into DNA in place of thymine, producing an incorporation error. 2. 2nd Replication: 5-bromouracil might mispair with guanine. 3. 3rd Replication: the guanine on the newly-synthesized strand from the previous replication pairs with cytosine, producing a permanent mutation. The other strand with 5-bromouracil can pair with guanine to produce another incorporation error or adenine where no replicated error occurs.
Explain how base excision repair repairs modified bases.
1. Glycosylases recognize and remove defective bases, producing an AP site. 2. AP endonuclease cleaves the phosphodiester bond next to the missing base and removes the rest of the nucleotide. 3. DNA polymerase fills in the gap and ligase seals the nick.
List the two types of double strand break repair.
1. Homologous recombination repair 2. Nonhomologous end joining
What are three ways X-rays can cause chromosome breakage?
1. Ion beams break phosphodiester bonds in DNA backbone 2. Damage bases 3. Point mutations
List two histone modifications used for eukaryotic regulation.
1. Methylation of tails of histones 2. Acetylation of tails of histones
Explain how mismatch repair repairs replication errors.
1. Mismatch repair proteins recognize/bind to the abnormal helical structure and identify the incorrect base. 2. Exonucleases remove the section of the new strand, which spans from the methylated sequence to the mismatch. 3. DNA polymerase fills in the gape and ligase seals the nick.
List the four effects of point mutations at the protein level.
1. Missense 2. Nonsense 3. Silent 4. Readthrough
What are two causes of spontaneous frameshift mutations? What do they both produce that leads to these mutations?
1. Template slippage 2. Unequal crossing over Insertions and deletions of one or more base pairs.
What happens to the lac operon when glucose levels are low?
1. cAMP levels are high and more likely to bind to CAP. The cAMP-CAP activator forms. 2. As a result, RNA polymerase is able to bind more efficiently to DNA. 3. When lactose (inducer) is present, high rates of transcription and translation of the structural genes occur. 4. Glucose is produced from lactose.
What happens to the lac operon when glucose is high?
1. cAMP levels are low and less likely to bind to CAP. 2. As a result, RNA polymerase can't efficiently bind to DNA, so little transcription occurs.
Draw/list the four components of the lac operon.
1. lacl: regulatory gene that codes for the repressor. 2. lacP: operon promoter that binds RNA polymerase to allow for transcription. 3. lacO: operatory that interacts with the repressor to inhibit transcription of the operon. 4. lacZ, lacY, lacA: structural genes that are transcribed and translated when the repressor is bound to the operator. lacZ: B-galactosidase lacY: permease lacA: transacetylase
Draw/list the five components of the Trp operon.
1. trp repressor: normally inactive repressor that can't bind to the operator without trytophan. 2. Promoter: operon promoter that binds RNA polymerase to allow for transcription. 3. Operator: interacts with the repressor to inhibit transcription of the operon. 4. Leader sequence: contains an attenuator that inhibits transcription of the operon with tryptophan present. 5. Structural genes: transcribed and translated when the repressor is bound to the operator.
To incorporate a base analog, how many rounds of replication are required? How many rounds of replication to obtain a complete transition are required?
1; 2
How many rounds of replication are needed for wobble base pairing to alter both strands of the DNA? What kind of mutation results from this spontaneous mutation?
2 rounds of replication; transition mutation
Ac element
A complete transposable element with a functional transposase and repeats.
Homologous recombination repair
A double strand break repair that uses the sister chromatid to repair the break in DNA.
Nonhomologous end joining
A double strand break repair that uses trans-lesion polymerases to bypass lesions on the DNA. Can lead to mutations.
Point mutation
A gene mutation in which a single base pair in DNA has been changed to another base pair.
Neutral missense
A missense mutation that doesn't affect protein function.
Missense
A point mutation changes the amino acid, which may alter protein function.
Nonsense
A point mutation changes the codon into a stop codon.
Readthrough
A point mutation changes the stop codon into a codon that codes for an amino acid, resulting in a longer protein.
Silent
A point mutation produces a codon that codes for the same amino acid.
How do riboswitches inhibit translation?
A regulatory protein binds to the riboswitch and stabilizes a secondary structure that masks the ribosome binding site. Translation is inhibited.
Operon
A segment of DNA containing controlling regions and structural genes controlled by those regions.
How can most cells have the same genetic content yet have different functions in the body?
Alternative 3' cleavage and splicing of pre-mRNA produces different proteins in different tissues/times in development based on the exons that are removed.
The lac operon produces three products: _________________, _______________, and __________________. These products then become ____________ that can be converted to _______________ and ______________.
B-galactosidase; permease; transacetylase Lactose Glucose; galactose
Wobble base pairing - Which bases can wobble pair and with how many hydrogen bonds?
Bases mispair due to flexibility in the DNA helix: - A and C can wobble pair with 2 hydrogen bonds. - T and G can wobble pair with 2 hydrogen bonds.
In what cells does proofreading occur during replication?
Both prokaryotes and eukaryotes.
Base analogs lead to what kind of mutation after two rounds of replication?
Complete transitions
Compare the transcriptional potential of condensed chromatin, decondensed chromatin, and naked DNA.
Condensed chromatin makes it harder for proteins to interact with the DNA. Decondensed chromatin and naked DNA makes DNA easier to access. Decondensed chromatin is DNase1-sensitive; the chromatin has DNaseI hypersensitive sites that allow DNase1 endonucleases to cleave the DNA and remove a nucleosome. This produces binding sites for regulatory proteins (facilitates transcription).
Define the following notations of the operator of the lac operon and whether each are trans dominant, trans recessive, or cis dominant: Oc
Constitutive operator; cis dominant
Direct repair of mutations
Corrects structure of abnormal nucleotide without replacing the nucleotide.
Regulatory elements
DNA sequences that aren't transcribed, but play a role in regulating other nucleotide sequences.
Transposable genetic elements
DNA sequences that can move between sites and chromosomes and can alter phenotypes as a result.
Define the following notations of the promoter of the lac operon: P-
Defective promoter
Ds element
Dissociation element that lacks the transposase gene due to deletion. Requires the Ac transposase to move.
Regulatory genes
Encode products that interact with other sequences and affect the transcription and/or translation of these sequences.
Structural genes
Encode proteins that are used in metabolism or play a structural role in the cell.
Oxidating agents lead to what kind of mutation after two rounds of replication?
GC to TA transversions
Explain regulation by attenuation in the trp operon when: - Tryptophan levels are high - Tryptophan levels are low
High: 1. As RNA polymerase transcribes the leader sequence, the ribosome translates the mRNA just behind it. 2. Tryptophan is readily available for the ribosome and the ribosome continues downstream, blocking region 2. 3. Region 2 is unable to pair with region 3, so region 3 and region 4 bind together. 4. Transcription is terminated. Low: 1. As RNA polymerase transcribes the leader sequence, the ribosome translates the mRNA just behind it. 2. Tryptophan is lacking. The ribosome stalls while the polymerase continues transcribing. 3. Regions 2 and 3 pair, continuing transcription.
Describe how acetylation and deacetylation of histones affect gene expression.
Histone deacetylation: Histone deacetylases remove the acetyl group from the histone, producing a positive charge on the histone tail. This tightens the association b/w histones and the DNA. Histone acetylation: Histone acetyl transferases transfer an acetyl group onto the histone, removing the positive charge on the histone tail. This loosens the DNA-histone association.
Define the following notations of the repressor of the lac operon and whether each are trans dominant, trans recessive, or cis dominant: I+ I- Is
I+: normal I-: defective repressor; trans recessive Is: super repressor; trans dominant
How does template slippage occur?
Intercalating molecules can slip between base pairs in DNA, distorting the helix and causing template slippage during replication.
What happens to the lac operon in the presence of lactose?
Lactose is converted into allolactose by B-galactosidase. Allolactose binds to the repressor, which undergoes an allosteric change in response. The operator binding site is also altered when the repressor binds to allolactose.
Explain how helix-turn-helix motifs bind DNA, including: - Location - Characteristics - Binding site at DNA
Location: - Bacterial regulatory proteins - Related motifs in eukaryotic proteins Characteristics: - 2 alpha helices Binding site: - One helix binds to a major groove in DNA - The other helix binds to a dimer in the DNA.
Explain how zinc finger motifs bind DNA, including: - Location - Characteristics - Binding site at DNA
Location: - Eukaryotic regulatory and other proteins Characteristics: - Loop of amino acids with zinc at base Binding site: - Major groove
Explain how leucine zipper motifs bind DNA, including: - Location - Characteristics - Binding site at DNA
Location: - Eukaryotic transcription factors Characteristics: - Helix of leucine and a basic arm - 2 leucines interdigitate (connect with each other like a zipper) Binding site: - Basic arms bind to 2 adjacent major grooves
Mutations can cause a loss or gain of function in proteins. Compare and contrast these two effects.
Loss of function: - Causes complete or partial absence of protein function - Recessive acting (require both alleles must break) Gain of function: - Causes cell to produce a protein that isn't normally present. - Dominant acting (only one allele to break)
Which DNA repair mechanism removes lesions?
Nucleotide Excision Repair
What kind of cistronic mRNA is produced when the structural genes of the lac operon are transcribed?
Polycistronic mRNA
What can UV light do to DNA?
Produce pyrimidine dimers in the DNA (mainly thymine dimers), which distort the helix and inhibit replication.
Transition point mutation
Purine to purine or pyrimidine to pyrimidine
Transversion point mutation
Purine to pyrimidine or vice versa
Positive control of transcription
Regulatory protein (activator) binds to DNA to stimulate transcription.
Negative control of transcription
Regulatory protein (repressor) binds to DNA to prevent transcription.
Anti-sense RNA
Small RNA molecules complementary to parts of the mRNA and inhibit translation.
Compare and contrast somatic vs. germline mutations.
Somatic mutations: - Occur in nonreproductive cells - Passed on to new cells via mitosis Germline mutations: - Occur in cells that give rise to gametes - Passed down from parent to offspring via meiosis and sexual reproduction - Offspring have mutation in ALL of their cells
Pyrimidine dimer
Structure in which covalent bonds form between two adjacent pyrimidine molecules on the same strand of DNA; disrupts normal hydrogen bonding between complementary bases and distorts the normal configuration of the DNA molecule.
What happens to transcription in negative inducible control? - When substrate is absent - When substrate is present
Substrate absent: transcription OFF Substrate present: transcription ON
What happens to transcription in positive inducible control? - When substrate is absent - When substrate is present
Substrate absent: transcription OFF Substrate present: transcription ON
What happens to transcription in negative repressible control? - When substrate is absent - When substrate is present
Substrate absent: transcription ON Substrate present: transcription OFF
What happens to transcription in positive repressible control? - When substrate is absent - When substrate is present
Substrate absent: transcription ON Substrate present: transcription OFF
Explain how transposons can cause mutations using the Ac-Ds system in maize.
The Ac-Ds system in maize can produce a yellow phenotype and a variegated phenotype. The yellow phenotype occurs when the dominant (purple) allele of a heterozygote is disrupted by the transposition of a Ds element into the allele. This occurs when the Ac element produces a transposase enzyme that move the Ds element into the allele. The variegated phenotype occurs similarly, but in some cells, the Ds element leaves the allele, producing a purple pigment. Cells wherein the Ds element doesn't leave produce a yellow pigment.
DNA methylation
The addition of methyl groups to bases of DNA by DNA methyl transferases after DNA synthesis; may serve as a long-term control of gene expression.
If newly-synthesized strands experience slippage (i.e., the strand loops out), what occurs?
The addition of one nucleotide on the same strand.
Histone code
The combination of modifications present that help regulate chromatin structure and transcription.
Constitutive gene expression
The gene is always expressed.
What is one way the genetic code differs from the histone code?
The genetic code depends on a particular codon, whereas the histone code is more complex as there are more modifications to be considered.
How is the old DNA strand distinguished from the new DNA strand during mismatch repair in E. coli?
The new strands are not yet methylated, while the old strands are methylated.
Explain why positive regulation of the lac operon is required.
The operon promoter is weak because the -10 and -35 consensus regions aren't ideal, inhibiting the binding of RNA polymerase to the promoter.
Catabolite repression
The process by which the presence of a preferred sugar (e.g., glucose) source inhibits the transcription of certain operons (e.g., lac operon).
What causes depurination?
The removal of a glycosidic bond (b/w a base and sugar).
If template strands experience slippage (i.e., the strand loops out), what occurs?
The removal of one nucleotide in the new strand.
When tryptophan levels are high, what happens to the trp operon?
The repressor binds to the operator and NO transcription occurs.
What happens to the lac operon in the absence of lactose?
The repressor binds to the operator and inhibits transcription.
When tryptophan levels are low, what happens to the trp operon?
The repressor can't bind to the operator and transcription occurs.
How do oxidating agents damage DNA?
They convert guanine into 8-oxyguanine, which pairs with adenine instead of cytosine during replication. This adenine may pair with a normal thymine during the next replication, causing GC to TA transversions.
Why is attenuation restricted to prokaryotes?
Transcription and translation occur simultaneously in prokaryotes. In eukaryotes, these processes occur in different places.
Inducible control of transcription
Transcription is normally off and turned on when a small molecule binds the regulatory protein.
Repressible control of transcription
Transcription is normally on and is turned off when a small molecule binds the regulatory protein.
How do anti-sense RNA's inhibit translation?
Under certain conditions, a regulatory gene is activated and transcribed into an anti-sense RNA. The anti-sense RNA pairs with the target mRNA and blocks the ribosome-binding site, inhibiting translation.
Explain how prokaryotes utilize direct repair of pyrimidine dimers.
Use an enzyme, photolyase, that absorbs light and cleaves the covalent bonds in the dimer.
How does DNA methylation regulate gene expression?
Usually represses gene expression; Methylated CpG sequences attracts deacetylases, which repress transcription due to the removal of acetyl groups from histone tails.
Regulated gene expression
When expression of a gene changes in response to any metabolic, environmental or pharmacological intervention.
When do X-rays cause the most damage in dividing cells?
When the chromosomes are condensed in mitosis.
After two rounds of replication, depurination leads to...
a permanent mutation.
5-bromouracil is a base analog that normally pairs with _____________, but can also pair with _____________.
adenine; guanine
2-aminopurine is a base analog that normally pairs with _____________, but can also pair with _____________.
thymine; cytosine
