DNA To Proteins (Gene Expression) Flashcards
What are mutagens? What are the major food mutagens? Provide examples
Mutagens- anything that causes a mutation in cell (changes DNA)
Food mutagens are heterocyclic amines (food cooked at high temps, above 450 degree F), Flavonoids (plants), MOLD and other compounds formed due to FOOD Preservation (pickling)
Describe the purpose of the Ames test and example of compounds involved.
Ames test- standard test in biotechnology, used to test in bacteria whether a given chemical can cause mutation in DNA of tested organism.
-reveals mutagenic potential of compounds by reverting histidine-auxotrophic phenotype of S. Typhimurium mutant (salmonella) (revert mutations in mutant organism)
What are the different sources of DNA Damage? When is DNA most vulnerable and Why? What occurs as a result of DNA Damage?
Exogenous (external) sources- chemicals, radiation
Endogenous(internal) sources- ROS (reactive oxygen species) replication errors, spontaneous hydrolysis
DNA most vulnerable during REPLICATION
as a result of DNA damage, mutations in somatic cells and germline cells occur, which lead to cancer, defects in cellular functions, cell death and senescence
Aging- leads to decline of efficiency and accuracy of DNA repair,
List the different types of DNA Damage and how they work?
- Oxidation- most frequent form of DNA damage, seen in aging
- Alkylation- addition of alkyl groups to the bases (methlyation is the most common one)
- Deamination- loss or substitution of amino groups at the bases.
- Dupurination/Depyrimidination- loss of bases at nucleoside residues
- Formation of Base Dimers- thymine and cytosine dimers and more complex heterocycles induced by ionizing radiation and carcinogens (tobacco smoke)
- Single and double stranded DNA breaks
- Mismatch- (replication error)
Explain what occurs in Deamination, including its frequency and preservatives that are involved. What are the roles of Nitrous acid and Bisulfite? Does deamination occur faster in single-stranded or double stranded DNA?
Deamination (spontaneous hydrolysis rxn)- converting Cytosine to Uracil by releasing ammonium.
C to U conversion occurs at frequency of 1 per 10^7 bases in 24 hours (100 per day in mammals)
Deamination 100 times faster in ssDNA (single-stranded) than DsDNA
Nitrous Acid formed from organic precursors, nitrosamines, nitrite and nitrate salts, is an accelerator of deamination bases.
Both Nitrous Acid and Bisulfite used as preservatives.
What is the benefit of DNA having Thymine instead of Uracil?
It protects DNA from losing its bases.
Establishing thymine as one of 4 bases in DNA is crucial turning point in evolution, makes long term storage of information possible.
In RNA, accumulation of unwanted Uracils cannot be distinguished from natural Uracils. Whereas in DNA, uracil can easily be recognized as foreign and corrected or repaired (replace with cytosine).
Explain the process of depurination and what is created as a result of it. Also describe the frequency of depurination and its effect on RNA.
Depurination- hydrolysis of N-glycosidic bond between base and pentose, creating lesion called ABASIC site (sugar-phosphate chain)
Process occurs at higher rate for purines (A, G) than pyrimidines (C, T).
One in 10^5 purines are lost in every mammalian cell over 24 hours.
The depurination of ribonucleotides and RNA is much slower and not considered physiologically significant.
What does ionizing radiation do ? What is the most common type of it? Explain and provide an example of how this radiation affects the cell.
Ionization energy-It is significant source of DNA damage that causes various DNA modifications depending on radiation energy.
UV light- most common type of ionizing radiation
It will induce condensation of 2 ethylene groups into CYCLOBUTANE Ring, which can form in cell between 2 adjacent pyrimidine bases, like THYMINES.
In humans, UV and ionizing radiation- 10% of all DNA damage caused by environmental agents, can result in SKIN CANCER.
Describe the most common forms of Alkylation of DNA and examples of mistaken DNA Alkylation. What is role of alkylating agents ?(include examples).
Addition of methyl group to Guanine to yield O^6-METHYLGUANINE.
Mistaken DNA ALkylation -caused by alkylating agents that are normally present in the cell , like S-ADENOSYL_METHIONE (donor of methyl group for intracellular reactions)
Mistaken alkylation caused by toxins called alkylating agents. Ex: NITROGEN MUSTARD
Nitrogen mustard and other alkylating agents (Cisplatin) are powerful CHEMOTHERAPEUTIC ANTICANCER Drugs (alkylate guanine base, preventing double helix stands from staying attached to each other, prevent multiplying of cancer cells).
What happens when Guanine is methylated? What does O^6 methylguanine interact with? What can it not interact with? Provide the 2 important things in base-pairing.
Methylation allows guanine to form a pair with Thymine in DNA.
O^6 methylguanine cannot base pair with cytosine. It pairs with thymine.
Base pairing- H-bond formation and correct matching geometry of bases are important.
How does the formation of O^6 methylguanine result in inherited mutation?
O^6methlyguanine cannot base pair with cytosine, instead it pairs with Thymine.
During replication, T is incorporated against methylguanine. A second replication will occur and the previously incorporated T will be paired with A, forming G-C to A-T mutations.
What is the most frequent source of mutagenic alterations in DNA? What kind of products will result from ionizing irradiation? What is the most frequently detected product of DNA Oxidation?
What are the uses of this product and where does it accumulate?
DNA Oxidative Damage
REACTIVE OXYGEN SPECIES (ROS) like Hydrogen peroxide, hydroxyl and superoxide radicals arise during ionizing irradiation and as byproducts of oxidative metabolism.
Most frequently detected product of DNA oxidation is 8-OXO -2’DEOXYGUANOSINE (8-OXO-G).
accumulation of 8-OXO-G used to measure rate of oxidative stress in cells and tissues.
it accumulates in nuclear and mitochondrial DNA with AGING.
Describe the level of mistakes made during replication with everyday activities compared to replication with proofreading and mismatch repair.
DNA mismatch repair corrects about 99% of replication errors, which increase accuracy to one mistake per 10^9 nucleotides copied. This is higher than all of the mistakes made in daily activities (professional typer, airline luggage, driving a car in US) and DNA replication without proofreading (1 mistake per 10^5 nucleotides), replication with proofreading (1 mistake per 10^7 nucleotides copied).
What is the first line of defense against unwanted changes in DNA? Elaborate more on how the use of genes play of role in this line of defense.
Redundancy of genome. Many genes exist in multiple copies and partake in redundant metabolic pathways that can substitute one another.
What is a codon? How does it affect mutations?
Triplets of nucleotides encoded for an amino acid, so
many mutations are SILENT because of base Substitutions that do not change the amino acid (ex: codon change from AAA to AAG is still same aa lysine
What is diploid genome? provide an example
Diploid genome carries two alleles of the same gene
ex: sickle cell anemia (2 copies of HbS allele for sickle)
elaborate on how the structure of DNA serves a role in redundant metabolic pathway.
what is this an example of?
What protects genetic information in DNA?
DNA has two complementary strands (besides parental strands), that each carry the same information.
The last two redundancies (copied strands), allow to restore genetic information if one of the copies is damaged (ex: homologous recombination)
Base pairing and structuring of DNA protects genetic information which is base-encoded.
What are the different types of DNA repair?
What are characteristics of repair systems?
- Base excision repair
- Nucleotide excision repair
- Mismatch repair
- Homologous recombination
- Non-homologous end joining
repair systems are redundant, and recognize similar DNA errors and overlap in spectra of damages they repair.
Most of them fix both intrinsic and extrinsic damages.
What are the 3 steps of DNA repair that all repair pathways work by?
- segment of damaged strand excised
- repair DNA Polymerase fills in missing nucleotide in strand that was excised using other strand as template.
- DNA ligase seals nick (requires ATP hydrolysis)
Explain the repair mechanism with Mismatch repair
When does mismatch repair not applicable?
proteins of mismatch repair complex recognizes unpaired ("melted") part of double-stranded DNA as both nucleotides are "natural"
Then, METHYLATION of DNA by specific METHYLASES will distinguish the "old strand" from new one
Mismatch repair (single stranded DNA break)does not work when there are double stranded DNA breaks(error in both strands of helix).Explain the 2 strategies that double-stranded DNA breaks require for repair, and compare and contrast which of the strategies are used in eukaryotes and prokaryotes, provide examples.
For ds-DNA, they require strategy of Nonhomologous end joining or Homologous recombination.
Nonhomologous end joining - specially developed in prokaryotes
Ex: Bacteria DEINOCOCCUS RADIODURANS survive multiple radiation induced double stranded DNA breaks by efficiently repairing them.
nonhomologous end joining also important in eukaryotes. Eukaryotes undergo homologous recombination as well (better strategy for them)
Describe the mechanism for nonhomologous end joining repair and a potential consequence of this action
- Nonhomologous end joining- process begins with nuclease processing DNA end (chewing broken parts), DNA ligase then joining the two broken parts together (may result in loss of nucleotides at repair site)
Explain the mechanism for Homologous Recombination
- Homologous recombination-
process - recombination specific nuclease will digest 5’ ends of broken strands
- broken strand will be invaded by complementary base pairing
- Repair polymerase uses undamaged complementary DNA as template (3 ‘ ends become longer, 5’ end extended by DNA polm).
- Invading strand released and complementary base pairing allows broken helix to re-form
- DNA synthesis continues using complementary strands from damaged DNA as template
6, DNA ligase sticks back pieces together.
Which strategy is preferred for double stranded DNA repair?
When does homologous recombination occur?
Homologous recombination is more preferred because after repair, you do not Lose nucleotides. Whereas in Nonhomologous end joining- possible to lose nucleotides at end of process
Homologous recombination occurs after DNA has been duplicated, before cell division (chromosomes separated)
What is the role of RNA mismatch repair system?
It removes replication errors that escape PROOFREADING driven by DNA polymerase.
What is preserved in genome sequences?
Differentiate between conserved or invariant nucleotide sequences and variable sequences.
A record of the FIDELITY of DNA replication and repair
Even after 100 million years later, faithfulness of replication and repair allow nucleotide sequences of DNA both whale and humans to still remain closely related (only 4 nucleotide differences).
Conserved (invariant) sequences- those that encode for important genes or traits preserved in evolution
Variable- regions that encode for obsolete functions. The number and type of nucleotide substitutions accumulated over time serve as EVOLUTIONARY CLOCK
To determine when species diverged from common ancestor .
What is the driving force of evolution? Why are mutations beneficial in certain environments?
DNA mutagenesis is the driving force of evolution.
mutations that appear harmful nevertheless been preserved in human population, because in certain conditions they may be advantageous.
ex: frequencies of SICKLE CELL CARRIERS are high in MALARIA- endemic areas (sickle cell allele protects them from getting malaria)
sickle cell anemia- single nucleotide change (mutation)
How does the DNA Repair system change over the years of one’s life?
DNA repair system not evolved to protect individuals after reproductive age (DNA stops proofreading DNA’s)
incidence of colon cancer or other cancers increase as one ages.
What is Central Dogma and who coined the term?
central dogma- flow of genetic information from DNA to RNA to protein.
term coined by Crick
Explain how gene readout begins, including formation of RNA
portions of genomic DNA are transcribed into RNA that is complementary to either one of the DNA strands.
Ex: only one strand among two encodes for RNA in each gene (can be either one of them).
some genes need a few transcription factors to acquire proteins, while others need many factors (actin)
What are the major differences between RNA and DNA ?
Sugar difference and Base difference
RNA- uses ribose sugar (2 OH in ring), and has Uracil instead of T nucleotide.
DNA- uses deoxyribose sugar and has Thymine instead of Uracil
Despite differences, Ribonucleotides in RNA are connected to double helix in similar manner as deoxyribonucleotides.
They both also, have 5’ and 3’ ends.
RNA and DNA are both have 5’ to 3’ orientation extension of RNA- 5’ to 3’ direction, each nucleotide added to 3’end which extends.
During transcription, how is the RNA strand complementary to one of DNA strands?
What type of orientation is DNA and new RNA strand?
What is a coding strand?
The DNA strand that RNA copies is built on creating matching A-U and G-C interactions between RNA and bases and DNA bases (template strand)
same complementary interaction between RNA and DNA strands as DNA in helix.
RNA and template DNA strand are in ANTIPARALLEL orientation
Coding strand- opposite strand from template strand in double-stranded DNA helix. This strand has Ts (instead of U’s and its it has same 5’ to 3’ orientation as RNA, and the sequence shows what gene encodes.
Explain what occurs with newly synthesized RNA regarding complementary interactions and why this occurs.
newly synthesized RNA is released from complex with DNA form secondary structure, bases seek complementary interactions for thermodynamic stability to occur.
RNA is single stranded and will seek stability by forming base pairs between bases on . same strand dna being double-stranded bases on opposite sides can form compl interactions r RNA copies are so form stem loop and secondary structures
Differentiate between interactions in Double stranded vs single stranded DNA and how it differs from complementary interactions in RNA.
single stranded nucleic acids (DNA or RNA)- stability is sought through base pairs between bases on SAME strand.
In double-stranded (like DNA) only, bases from OPPOSITE strands in double helix form compl. interactions.
Whereas, RNA copies are single stranded- and form STEM-LOOPS and other complex 3D SECONDARY structures by forming internal Watson-Crick pairs A-U, and G-C
What are the ways RNA secondary structures are stabilized?
By internal base-pairing, canonical and non-canonical Watson-Crick pairing.
compare and contrast Canonical and Non-canonical Watson-Crick base pairing that help stabilize RNA secondary structures
Non-conventional (non-canonical) pairs like G-A, C-U
have lower number of H-bonds and less optimal geometry than Canonical Watson-Crick pairing- G- C, A- U
in absence of canonical, non-canonical will form, stabilizing RNA 3D strcutures.
Describe the structure and folding of non-coding RNAs like tRNAs, rRNAs.
tRNAs, rRNAs and other non-coding RNAs are normally heavily structured
folding of molecules- temperature dependent, can be predicted by computer modeling with higher probability than folding patterns of proteins.
Explain how RNA secondary structures form ribozymes and ribonucleoprotein complexes.
multifaceted folding patterns like RNA double helix create surface for proteins to bind in RNA protein complexes like Ribosomes
also the RNA secondary structures may work as catalysts of biochemical reactions- Ribozymes (RNA-based enzymes). Ribozymes have been abundant in RNA world billions of years ago before proteins involved.
Describe the structure of 70S ribosome
16S, 23S, and 5S rRNAs are on outside of ribosome (colored cyan, gray, gray-blue); small and large ribosomal proteins overlap rRNAs, ribosome. While, tRNAs (yellow, orange) and MRNA (green) are furthest inside ribosome.
Explain how RNA is synthesized, including enzymes and other structures involved.
RNAs synthesized by enzymes called RNA polymerases.
The RNA polymerase has to separate (melt) the strands first and make one strand available for readout.
Polymerase then moves along double strand in direction of template strand (DNA) in from 3’ to 5’ direction, ensuring RNA as antiparallel strand is made in 5’ to 3’ direction.
first incoming nucleotide- unused 5’ triphosphate, designate 5’end. Next ribonucleoside triphosphate forms phosphodiester bond and provides phosphate to available 3’ end.
How is the Transcription complex stabilized by the RNA-DNA hybrid? Explain why RNA-DNA hybrid is more stable than corresponding DNA-DNA hyrbrid in ds-DNA?
during transcription, melting “window” moves along the template; this window is where RNA polymerase maintains RNA-DNA hybrid.
RNA-DNA hybrid is thermodynamically more stable than DNA-DNA hybrid, since it provides great stability to moving transcription machinery that does not dissociate form DNA when it stops
What allows RNA transcript to emerge from complex?
What are RNA polymerases made of and what is the speed?
RNA polymerase will actively displace RNA from the hybrid using specialized structures.
RNA polymerases made of single polypeptide (despite complex process)
RNA polymerases have a speed of about 200 nucleotides per second and error rate of one misincorpration per 10^4 nucleotides.
What types of RNAs do prokaryotes and Eukaryotes share? What structures do eukaryotes have, that prokaryotes don’t have?
They share mRNAs, tRNAs, and rRNAss.
eukaryotes have non-coding regulatory rRNAs and RIBOZYMES
Differentiate between the number of RNA polymerases eukaryotes have vs prokaryotes
Prokaryotes have one type of RNA polymerase
Eukaryotes- MANY different types of RNA polymerases (I , II, and III) that each synthesize a type of RNA (mRNAs, tRNAs, and rRNAs).
List the various types of RNA and their functions.
- Messenger RNAS (mRNA)- code for proteins
- Ribosomal RNAs (rRNA)- form core of ribosome’s structure and catalyze protein synthesis
- microRNAs (miRNAs)- regulate gene expressoin
- transfer RNAS (tRNAs)- serve as adaptors between mRNA and amino acids during protein synthesis
- other non-coding RNAs- used in RNA splicing, gene regulation, telomere maintenance, and other processes.
How does released genetic info become greatly amplified on transcription level?
Transcribing genes have a number of RNA polymerase molecules reading the information and producing RNA one after another, reading DNA and following each other head to tail.
Bumping into each other improves transcription rate by pushing RNA polymerase off the transcription start site.
rate of transcription varies for different genes. Stages of gene expression are coordinated.
What must occur for initiation of transcription? Explain the paradox of transcription in prokaryotes, and the major structure that solves this paradox.
RNA polymerase must bind to specific DNA sequence called PROMOTER.
Paradox of transcription: same enzyme should have high affinity to particular DNA sequence at specific place. at same time, the same enzyme must be tightly bound to DNA during elongation of transcription regardless of sequence.
Sigma factors solves paradox of transcription.
