DNA To Proteins (Gene Expression) Flashcards
(118 cards)
1
Q
What are mutagens? What are the major food mutagens? Provide examples
A
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)
2
Q
Describe the purpose of the Ames test and example of compounds involved.
A
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)
3
Q
What are the different sources of DNA Damage? When is DNA most vulnerable and Why? What occurs as a result of DNA Damage?
A
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,
4
Q
List the different types of DNA Damage and how they work?
A
- 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)
5
Q
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?
A
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.
6
Q
What is the benefit of DNA having Thymine instead of Uracil?
A
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).
7
Q
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.
A
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.
8
Q
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.
A
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.
9
Q
Describe the most common forms of Alkylation of DNA and examples of mistaken DNA Alkylation. What is role of alkylating agents ?(include examples).
A
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).
10
Q
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.
A
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.
11
Q
How does the formation of O^6 methylguanine result in inherited mutation?
A
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.
12
Q
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?
A
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.
13
Q
Describe the level of mistakes made during replication with everyday activities compared to replication with proofreading and mismatch repair.
A
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).
14
Q
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.
A
Redundancy of genome. Many genes exist in multiple copies and partake in redundant metabolic pathways that can substitute one another.
15
Q
What is a codon? How does it affect mutations?
A
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
16
Q
What is diploid genome? provide an example
A
Diploid genome carries two alleles of the same gene
ex: sickle cell anemia (2 copies of HbS allele for sickle)
17
Q
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?
A
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.
18
Q
What are the different types of DNA repair?
What are characteristics of repair systems?
A
- 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.
19
Q
What are the 3 steps of DNA repair that all repair pathways work by?
A
- 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)
20
Q
Explain the repair mechanism with Mismatch repair
When does mismatch repair not applicable?
A
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).
21
Q
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.
A
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)
22
Q
Describe the mechanism for nonhomologous end joining repair and a potential consequence of this action
A
- 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)
23
Q
Explain the mechanism for Homologous Recombination
A
- 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.
24
Q
Which strategy is preferred for double stranded DNA repair?
When does homologous recombination occur?
A
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)
25
What is the role of RNA mismatch repair system?
It removes replication errors that escape PROOFREADING driven by DNA polymerase.
26
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 .
27
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)
28
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.
29
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
30
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)
31
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.
32
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.
33
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
34
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
35
What are the ways RNA secondary structures are stabilized?
By internal base-pairing, canonical and non-canonical Watson-Crick pairing.
36
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.
37
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.
38
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.
39
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.
40
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.
41
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
42
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.
43
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
44
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).
45
List the various types of RNA and their functions.
1. Messenger RNAS (mRNA)- code for proteins
2. Ribosomal RNAs (rRNA)- form core of ribosome's structure and catalyze protein synthesis
3. microRNAs (miRNAs)- regulate gene expressoin
4. transfer RNAS (tRNAs)- serve as adaptors between mRNA and amino acids during protein synthesis
5. other non-coding RNAs- used in RNA splicing, gene regulation, telomere maintenance, and other processes.
46
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.
47
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.
48
Why is the sigma factor( prokaryotic transcription) not released in the first attempt?
Normally, the RNA polymerase will make many attempts to escape promoter by synthesizing short ABORTIVE RNA PRODUCTS (eventually release sigma)
49
describe the process of transcription in prokaryotes.
1. RNA polymerase contains sigma factor that will recognize promoter of gene.
2. once transcription begins, sigma factor will be released
3. Polymerase moves forward and continues synthesizing RNA
4. Elongation continues until polymerase reaches terminating region called terminator (red)
5. Enzyme halts and gets released, as well as RNA transcript completed and released
Later on, polymerase will find another free sigma factor who will search for another promoter sequence.
50
Where exactly is the start site, that DNA polymerase must recognize DNA sequences to start RNA synthesis.
The start site, where the first RNA nucleotide is added is at +1 (after +10).
sigma factor recognizes -10 and -3 elements in promoter
51
Why is the consensus -10 and -35 elements similar in all bacterial promoters? What happens if there is a mutation in these regions? if mutation in nucleotides between consensus regions?
These elements are similar because the same enzyme binds them regardless of what RNA they transcribe.
mutation in consensus regions- promoter strength affected
mutation between consensus regions- NOT affected by the mutations as long at length between -10 and -35 stays the same.
since the nucleotides in between region can vary (can be changed), promoter strength will not be affected length can be affected)
52
How do Sigma factors allow RNA polymerase to synthesize specific RNAs?
What occurs during transcription in critical situations?
why might this be a strategy for bacterial cells?
different sigma factors (in bacterial cells) allow switching transcription from housekeeping genes to set of specialize genes using same RNA polymerase core enzyme.
Ex: sigma 70- housekeeping (yellow) , sigma 32- heat shock (red), sigma 38- starvation (green) sigma 54- nitrogen deprivation (blue)
in critical situations, number of genes must be turned off, while transcriptions of others are initiated, so cell can respond fast and make necessary proteins.
used in bacterial cells since they depend more on environment than eukaryotes (if bacteria cannot support temps, it cannot store nutrients), hence bacterial transcripts are short-lived.
53
What role does the terminator play in prokaryotic transcription? What occurs due to the action of terminator
Terminator-sequence that encodes STEM-LOOP and POLYU tract (help stop transcription) Once polymerase encounters multiple Us, RNA-DNA hybrid loosens and polymerase may pause and backtrack. Backtracking, polymerase may encounter stem loop that makes transcription complex fall apart, release RNA Product.
54
What is the most regulated process in the cell?
Transcription
55
How many consensus sequences do eukaryotes have and what are they? What recognizes and binds to specific consensus sequences in eukaryotes?
There are 4 consensus sequences in Eukaryotes:
-35, -30, (TATA box) transcription start site and +30.
Transcription factors recognize the consensus sequences.
56
Differentiate between eukaryotes and prokaryotes, in terms of number of RNA polymerases and function.
Prokaryotes- has only one RNA polymerase, where polymerase core machine is the same, but SIGMA SUBUNITS are different.
Eukaryotes- have 3 enzymes as DESIGNATED POLYMERASES:
- DNA polymerase I- transcribe most rRNA genes
-DNA polymerase II- transcribe protein coding genes and genes for other non-coding rRNAs (spliceosome)
-DNA polymerase III- transcribe tRNA genes, 5s rRNA gene , and small RNAS.
57
explain how different genes (on same chromosome) can be expressed by using either of the strands as template In what direction is RNA Synthesized and how should consensus sequences be positioned?
Genes with same DNA strand as template are always transcribed in same direction
genes that utilize opposite strands of DNA as template strands are transcribed in opposite direction (example, gene a and gene b)
RNA always synthesized in 5' to 3' direction. so DNA template should be in 3' to 5' direction. RNA should start with 5' end and 3' will extend. consensus sequences should be positioned so, -35 is on the left and -10 is further right as RNA synthesis goes from left to right.
both strands of DNA required for promoter and RNA polymerase to recognize it; template strand (DNA) downstream of promoter. other strand is coding.
58
Explain how transcription factors help initiate transcription in eukaryotes.
Transcription factors assist in bending of DNA that facilitates base exposure.
TBP (TATA- binding protein) inserts aromatic amino acid radicals (PHENYLALNINES) between base pairs and changes confirmation, so residues will work as levers to bend DNA (kink) at the promoter many of the factors that initiate transcription work like this. Ex: SAC7D from ARCHAEA
Later ,other transcription factors come along and assembly into active transcription complex with core RNA polymerase II.
59
what are the steps of eukaryotic transcription?
1. eukarotic promoter sequence contains TATA box that is recognized by subunit TBP on transcription factor TFIID.
2. TBP binds to TATA box sequence, creating distortion or bending of DNA.
3. once TFIID binds to promoter (TATA box), enables binding of TFIIB to region
4. rest of transcription factors and DNA polymerase form complex and they assemble at promoter
5. TFIIH pries apart double helix, and exposes template strand using ATP hydrolysis, and also phosphorylates RNA Polymerase II, releasing polymerase, starting transcription.
60
what are the most important steps in eukaryotic mRNA processing?
CAPPING, POLYADENATION, and SPLICING
61
Differentiate between the mRNA life of prokaryotes and eukaryotes, including the function of RNAases
Prokaryotic mRNA is short lived; they constantly degrade and get synthesized (bacteria-short life cycle of 3 mins)
Eukaryotic mRNA long-lived and may support expression of proteins in tissues for long period of time (0.5-10 hours)
RNAses -RNA-degrading enzymes that ensure RNA copies are always fresh and do not have unwanted modifications.
Fresh eukaryotic mRNA copies are protected through being capped, polyadenylated and spliced (unlike prokaryotes)
62
Explain the different functions of Capping, Polyadenylation, and Untranslated regions, including where they occur on the helical strands and when they occur.
Capping- covalent attachment of 7-METHYLGUANOSINE to 5' end triphosphate. It begins after RNA transcript emerges from RNA polymerase (25 nucleotides). Capping protects 5' end of transcript and signal for ribosome recognition
Polyadenylation- Poly A Polymerase adds POLY A- TAIL at 3'end in non-template manner. Before poly A-tail added, 3' untranslated region (3' UTR) is trimmed.
untranslated regions 5'UTR and 3'UTR - play protective and regulatory role in both eukaryotic and prokaryotic RNAs (translated region surrounded by untranslated region)
63
Explain the process of splicing that occurs in eukaryotes. Does splicing occur in prokaryotes?
What is the OPEN READING FRAME?
Splicing- process of cutting introns out and splicing exons together.
protein-coding genes are interrupted by non-coding sequences called INTRONS. Coding regions- exons.
Open reading frame (ORF)- nucleotide sequence that can be translated directly into polypeptide sequence, through set of codons.
To restore ORF- immature, newly synthesized mRNA (pre-mRNA) will get introns cut out and exons fused together.
NO splicing in prokaryotes
64
compare and contrast length of sequences of introns and exons and explain how this affects sequence.
Intron (non-coding) sequences are much LONGER than exons.
Long genes are prone to accumulate mutations.
most human genes broken into multiple exons and introns.
65
Explain how introns are ribozymes. Are all introns capable of self-splicing? if not, what may be used to aid splicing?
The borders between exons and introns are marked by specific sequences (GURAGU, YYYYYYYYNCAG)
Introns are ribozymes, as they can self fold into 3D secondary structures to bring nucleotides together in right position for self-splicing to occur.
No, only some introns can self-splice. other introns require assistance of SMALL NUCLEAR RNA (snRNA) and protein complexes (SnRNPS)
66
What is the advantage of alternative splicing? What percent of human gene does alternative splicing occur? Provide a major example of alternative splicing.
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provides evolutionary advantage, that increases variety of gene products (on same gene) without increasing mutagenesis level.
alternative splicing (exons can be skipped, but they MUST be in order)
95% of human genes
Ex of alternative splicing- generation of ANTIBODIES. Variable regions designed through alternative splicing, which provides a variety of candidate antibodies to recognize the antigen
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67
what are the only kind of mRNAs that can be exported out of the nucleus for translation and what do they exit out as?
Only mature, processed MRNAs can exit nucleus for translation. They will exit out as nucleoprotein complexes.
68
differentiate between how prokaryotes and Eukaryotes handle their mRNAs
Eukaryotes- all steps of mRNA processing occur simultaneously and start as soon as parts of mRNA emerge from transcription complex
Prokaryotes- translation of bacterial mRNAs may occur simultaneously with their synthesis as long as 5' end emerges.
69
When does does RNA splicing occur? What process can occur before or after splicing?
When is mRNA termed as pre-mRNA?
RNA splicing occurs after RNA has been transcribed and capped. Poly-Adenylation can occur before splicing or after.
Pre-mRNA or precursor mRNA is the immature mRNA that occurs before splicing or modification of 5' and 3' ends (Capping, poly-A)
70
How can SnRNP's (small nuclear ribonucleotides)
| help achieve faster alternative splicing?
Through complementary interactions between heavily structured and protein bound SnRNAs (snurps), the correct spatial orientation and thus completion of splicing maybe achieved faster.
RNA portion of snRNP base pairs with sequences that signal splicing, creating active site for splicesome
71
What are the steps in RNA splicing?
1. U1 (SnRNPs) recognizes 5' splice site and U2 recognizes lariat branching point site through complementary base pairing
2. U6 then rechecks 5' site by displacing U1 and base pairing with intron sequence itself (re-reading improves accuracy of splicing)
3. conformational changes in U2 and U6 due to ATP hydrolysis by spliceosome proteins, driving formation of active site.
4. after splicing, spliceosome dumps RNA binding proteins known as exon junction complex on mRNA to mark splicing complete.
72
Where does mRNA processing and RNA synthesis take place? where will mature RNAs be exported from? Where are mRNA molecules degraded to?
mRNA processing and RNA synthesis/processing take place in within nucleus.
mature eukaryotic mRNAs exit from nucleus.
and mRNA molecules are degraded in the cytosol.
73
What is translation?
synthesis of polypeptide chains by ribosomes that use mRNA as a template.
74
Which codons designate the start and stop sites? What makes stop sites different from start sites?
Translation starts with AUG (Met aa) and synthesis of polypeptide stops once ribosome reaches any of three stop codons (beginning with UA or UG).
stop codon does not encode any amino acids, while start codon (AUG) does encode aa Methionine.
75
What is the Opening reading frame (ORF)-?
ORF- linear sequence of codons that encode polypeptide sequence, from start codon to termination codon.
76
What is a codon?
sequence of three nucleotides (triplets) from 5' to 3' end that encode for amino acid.
77
Describe the components of genetic code and why it is referred to as degenerate or redundant?
There are 64 combinations of 4 nucleotides taken 3 at a time and only 20 amino acids, hence the genetic code is DEGENERATE (more than one CODON per amino acid) or REDUNDANT
78
Why does degeneracy of genetic code exist? Compare and contrast genetic code in eukaryotes vs prokaryotes.
degeneracy of genetic code results from mathematical probability to have just enough code variations for each amino acid out of 20.
also provides protection from mutations (substitutions of nucleotides silent , when 3rd nucleotide in codon substituted)
Both eukaryotes and prokaryotes have the same genetic code, but they differ in the FREQUENCY of codons in genome( for each particular aa, some codons may be more frequent than other).
79
In translation, what are the 2 most important non-coding RNA types?
tRNAs and rRNAs.
80
Explain what tRNAs are, and describe what an anticodon is.
tRNAs- short (80 nucleotide long) RNAs that are heavily structured and have exposed loop with ANTICODON.
Anticodon- triplet of nucleotides that is complementary to RNA codon in antiparallel orientation.
81
What is function of Aminoacyl tRNA synthetase (transferase)
enzyme that covalently attaches correct amino acid to 3'end of corresponding tRNA.
82
Explain the Wobble base pairing effect and why it occurs? How does the number of tRNAs and codons differ in humans?
Wobble base pairing effect is what allows there to be 61 possible codons (64 total - 3 stop codons) and species to have as few as 31 tRNAS . This occurs because structure allows for incomplete complementation between codon as long as first nucleotides are complementary (tRNA will recognize multiple codons for the aa it carries).
Humans have nearly 500 different tRNAs and only 48 codons.
83
Explain how the Aminoacyl-tRNA synthase creates a great energy source that can be used later in translation.
The energy of ATP hydrolysis used to create high-energy bond between acyl group of amino acid and tRNA is later used as energy source for synthesis of polypeptide bonds (translation)
84
Where does translation of all eukaryotic mRNAs occur? How does this translation location affect ribosomes?
Occurs in cytosol.
| depending on translation product destination, ribosomes may have different localization (Ex: ER or ribosome)
85
What are the largest ribonucleoprotein complex in the cells (also ribozymes)? What ribosomal processes occur in the nucleoli? What occurs before assembly of ribosomes and where does it occur?
Ribosomes are largest ribonucleoprotein complexes in cells.
synthesis of rRNA, their trimming and assembly of LARGE (3 rRNA)and SMALL (1 rRNA) ribosomal subunits occur in nucleoli.
Before ribosomes are assembled from subunits, they are exported from nucleus.
86
What are translation factors and how do they regulate translation on various cells?
Translation factors - large number of ribosomal proteins that permit recognition of RNA template and tRNA binding.
The three sites are A site- Aminoacyl-tRNA, P site- Peptidyl-tRNA and Exit site.
core proteins that form ribosomal subunits also regulate translation.
87
Describe how the initiation of translation occurs and the specific structures it requires.
What is the RBS in eukaryotes versus prokaryotes? Also distinguish between the small ribosomal subunits in two different cells and state it's role in translation.
Initiation of translation a multi-step process.
requires protein TRANSLATION INITATION FACTORS that recognize ribosome binding site.
Ribosome binding site- a specific nucleotide sequence that includes initiation codon ATG or AUG
that in case of prokaryotes, ribosomes assemble from 2 subunits)
The RBS in eukaryotes called KOZAK sequences
and RBS in prokaryotes called SHINE-DALGARNO sequence
small ribosomal subunit rRNAs has a sequence complementary to RBS (18s in eukaryotes; 16s in prokaryotes)
88
Explain the more complex process of initiation of translation in eukaryotes.
First, small ribosomal subunits recognizes cap at 5'end of mRNA (that may be distant from ATG codon). It will also recruit Met tRNA that recognizes non-coding ATG sequence. The complex will then scans RNA until it finds RBS and initiation codon. Once rbs found, transcription factor dissociate, large ribosomal subunit binds.
89
explain the process of the translation cycle and also discuss how translation is terminated.
Process of translation cycle:
1. charged tRNA carrying next aa to be added to polypeptide chain binds to A site on ribosome(base pairing with mRNA codon)
2. aa 3 (COOH group) in step 1 is uncoupled from tRNA at P site and joined by peptide bond to free amino group at A site
3. Large subunit shifts left, and move two bound tRNAs into E and P sites.
4. small subunits moves 3 nucleotides along mRNA molecules, bringing back to original position.
this movement ejects tRNA at E site and resets ribosome A site allow another charged tRNA to bind.
Translation is terminated when protein RELEASE FACTOR binds to termination codon and promotes dissociation of ribosomes on two recycle subunits.
90
Differentiate between mRNA encoding into polypeptides in eukaryotes vs prokaryotes and discuss where proteins are synthesized.
eukaryotes- single, mature mRNA encodes for a SINGLE polypeptide
prokaryotes- single mRNA may encode SEVERAL polypeptides with their own sites for translation initiation and termination
proteins are synthesized on polyribosomes.
91
Discuss how inhibition of bacterial transcription and translation is a powerful tool in fighting infection and what is used to inhibit these processes.
since, eukaryotic and prokaryotic ribosomes are very different, and translation is very complex, there are a large number of antibiotics that target bacterial translation (inhibit protein synthesis or RNA synthesis).
antibiotics:
Tetracycline- block binding of aminoacyl-tRNA to A site of ribosome
Streptomycin- prevent transition from initiation complex to chain elongation; also causes miscoding
Chloramphenicol- blocks peptidyl transferase reaction on ribosomes
Cyclohexamide- blocks translocation step in translation
Rifamycin- blocks initiation of transcription by binding to and inhibiting RNA Polymerase.
92
Discuss the type of post-translational protein modification that control activity (and 1/2 life) of translation products. Which protein marks other proteins for degradation.
Ubiquitin- small protein that marks other soluble cytosolic proteins
for degradation by PROTEASOME.
Proteasome- large protein-degrading complex, that utilize short lived and misfolded proteins.
Ubiquitin is added to target proteins by binding to LYSINE residues in POLYUBIQUITIN CHAINS
93
Explain the difference of various cell types in multiceullular orgnanisms?
What was the major breakthrough by studying differentiated cells.
They express different genes, produce different RNAs and proteins.
Understanding of how Dolly (cloned sheep) was made and IPSCs found through studying differentiated cells.
Major breakthrough was that animals can be cloned, IPSC (induced pluripotent stem cells)genome sequencing since DNA In all specialized cells types have the entire set of instructions needed to form an entire organism.
94
Describe the cloning experiment regarding mice and olfactory neurons.
Experiment helped find understanding about developmental potential of mature cells, like NEURONS(seemed immobile, very-specialized)
researchers (Rudolf J and Richard axel) sought to determine if mature olfactory neurons introduced to egg would revert to an undifferentiated state or could give rise to adult mouse with olfactory receptors.
theory worked, as resulting mice had organized odorant receptor that could not be distinguished from mice
95
Explain what IPSCS (induced pluripotent Stem cells) are, and how are they are made.
IPSCS (induced pluripotent stem cells) - cell that has potency to differentiate into multiple (plural) cell types.
IPSCS are made from artificial expression of 4 genes (*CMYC, oct 4, Sox2, Klf4) that each encode TRANSCRIPTION REGULATOR protein, that can reprogram any differentiated cell into pluripotent cell
IPSCs can proliferate in culture, have embryonic stem cell like properties, be stimulated by extracellular signals (growth, differentiating factors) to differentiate in almost any cell type in body.
96
what information can IPSCS's provide about cells?
IPSCs - info on clues to how development of late onset diseases may be triggered. compare patient specific cells differentiated from IPSCS with similar cells isolated from healthy subjects, watch how they respond to stress, environmental stimuli, treatment in vitro.
IT removes necessity of withdrawing cells or tissues using surgical methods, when most of original cells are dead like parkison's disease .
97
Name the steps at which gene expression can be regulated. Which regulation step is most paramount?
Steps that can be regulated:
when gene of how often it's transcribed, which RNA will be spliced or processed, which mRNAs exported form nucleus, how quickly mRNA are degraded, which mRNA translated into protein and how rapidly protein is degraded.
Regulation at TRANSCRIPTION level is paramount because it ensures no unnecessary intermediates are synthesized and cell resources not wasted.
98
*What is the most regulated process in the cell?
Transcription
99
How are genes arranged in bacterial chromosomes?
Bacterial genes are clustered and often expressed together.
100
How is transcriptional control exerted at beginning of transcription initiation? Also compare and contrast the regulatory sequences in eukaryotes vs prokaryotes.
Transcriptional control exerted by permitting or preventing of RNA polymerase at PROMOTER region.
in prokaryotes, regulatory sequence is small, several nucleotides in length and include one protein, (either transcription activator or transcription repressor).
Eukaryotes- regulatory region spans thousand of base pairs, include multiple proteins.
Transcription regulators bind to DNA as dimers.
101
Describe the specific DNA-binding elements of transcription regulators and the particular structure they form.
many transcription regulators that have structural elements capable of accessing DNA major groove along double helix.
Elements are tightly structured so distance between them aa residues that interact with base pairs are maintained like crisscross element like LEUCINE ZIPPER
In Leucine zipper, leucine residues alongside alpha helixes strengthen their binding through hydrophobic interacts, polar and charged aa at top of helical provide DNA sequence specific recognition.
102
what are the three examples of transcription activator elements and what similar roles do they show?
Zinc fingers, Helix-turn-helix motifs and Homeodomain (eukaryotes) are all transcription activators. They all orient recognition elements so that that they bind certain base pairs at promoter DNA. They have different structures, since they all recognize particular DNA sequences. their protein elements tightly fit to continuous elements at major groove (base pairs exposed for interaction)
103
Where is bacterial gene expression often organized? What are Operons?
In bacteria, gene expression organized in operons
Operons- set of genes expressed form a single promoter; operons are typical for bacteria since they are limited with energy sources and do not want to waste energy producing proteins that are not needed, cannot store large amount of polysaccharides .
104
Distinguish between different promoters used and what operators are and where they're located. What occurs in presence of repressors?
Promoters can be CONSTITUTIVE- work all the time as long as RNA polymerase available; or INDUCIBLE- have transcriptional switches that control operons, allow transcription from promoters at certain condition (allow cells respond well to environment).
Operators- regulatory elements that control activity of promoters by suppression. They are either adjacent to basic promoter that RNA binds polymerase binds to or overlaps with promoter.
Binding of REPRESSOR to operator by BLOCKING transcription.
105
What is the most complex molecular process in the cell?
Transcription regulation in eukaryotes- most complex process.
106
How is cell memory created?
Cell memory created by long-term transcription activators or repressors.
107
Explain how histone and DNA modifications can be inherited by daughter chromosome
The histone modification pattern may be inherited by daughter cells since one strand comes from parent cell with its own modification pattern . pattern may confer with same modification (epigenetic inheritance)
108
Elaborate on the reason why gene activation is more complex in eukaryotic cells and what components are included/needed.
transcription regulation of eukaryotes is most complex process, as they need assembly of multiple core (basic) proteins, tissue specific TRANSCRIPTION FACTORS at promoter, and distant DNA elements called ENHANCERS that recruit activator proteins and permit transcription.
eukaryotes can coordinate expression of certain genes by bringing together RNA loops from different part of chromosomes.
109
Explain how gene activity can be switched by combination of activator and repressor proteins. What is Lac operon and cAMP? What occurs in lac operon with availability of glucose? absence of glucose? Lactose present? Lactose absent?
Lac operon (E. coli) control set of genes that utilize lactose by breaking it down to galactose and glucose when available from extracellular sources.
glucose available- cell prefers glucose, turns lac operon off.
absence of glucose- bacteria makes cAMP which allosterically activates CAP, a lac operon activator that activates number of genes and allow bacteria to use alternative sources- lactose.
absence of lactose- lac operon specific repressor binds operator and turns operon OFF
lactose presence- byproduct ALLOLACTOSE bind repressor and remove transcription block. Lack of block will not ensure productive transcription, as activator is still required.
110
What are the regulatory noncoding RNAs?
miRNA (microRNAs), Small interfering RNA (siRNAs), and long non-coding RNAs.
111
Explain how gene expression can be controlled,(bacteria). What are two methods of regulating translation?
Gene expression can be controlled by regulating translation.
Sequence specific binding repressor protein can repress translation of some mRNAS by preventing the ribosome from binding to ribosome binding site (RBS) on mRNA through stabilization of double stranded structures (STEM loop) on ribosome binding site.
This will then make RBS inaccessible to ribosome assembly (it requires single-stranded, unfolded mRNA).
Stem loop structures can work alone (no accessory protein) as temp-dependent repressor of translation. The stability of A-T rich stem loops can change in rang of temps close human body temp (this occurs in translation-controlling elements in mRNA of parasites).
Increase in temperature, will separate DNA strands of stem loop (now single stranded, unfolded) and expose ribosome binding site, leading activation of translation, proteins made
112
Explain how gene expression can be controlled,(bacteria). What are two methods of regulating translations and describe what a stem loop is and how it regulates translation?
Gene expression can be controlled by regulating translation.
Sequence specific binding repressor protein can repress translation of some mRNAS by preventing the ribosome from binding to ribosome binding site (RBS) on mRNA through stabilization of double stranded structures (STEM loop) on ribosome binding site.
This will then make RBS inaccessible to ribosome assembly (it requires single-stranded, unfolded mRNA).
Stem loop structures can work alone (no accessory protein) as temp-dependent repressor of translation. The stability of A-T rich stem loops can change in rang of temps close human body temp (this occurs in translation-controlling elements in mRNA of parasites).
Increase in temperature, will separate DNA strands of stem loop (now single stranded, unfolded) and expose ribosome binding site, leading activation of translation, proteins made
113
List all of the non-coding RNAs.
tRNAs, rRNAs, spilcing-associated snRNAS, other non-coding RNA's associated with ribozymes like telomerase, microRNAs, Small RNAs, Long non-coding RNAs.
114
What are the functions of microRNAS (miRNAS)? How do they do these functions?
They direct the destruction of target mRNA in eukaryotic cells.
miRNAs are normally synthesized in eukaryotic cells and regulate gene expression by targeting complementary RNA molecules for destruction.
process:
MiRNAs form long secondary double-stranded structures that are recognized by RISC (RNA-induced silencing complex) proteins
RISC uses a single-stranded RNA fragment of miRNA and searches for complementary mRNAs that will match the miRNA and form double stranded element, leading to degradation.
This negative regulation of transcription is called silencing (controls gene expression on level of mRNA stability).
115
What are the 2 possible scenarios for microRNAs targeting compl. RNA for degradation?
scenario 1 : RISC with single stranded miRNA has found extensive, great match of complementary RNA leading to degrading of newly formed double-strand structure by nuclease within RISC.
scenario 2: RISC with single-stranded miRNA found less extensive, shorter match of complementary mRNA, leading to translation being reduced, mRNA sequestered and later degraded in cytosol
116
What is the functions of small interference RNA (siRNAS) ? How is their mechanism similar to miRNA?
Small interference RNAs (siRNAS) can be produced in response to foreign double-stranded RNAs.
They protect cells from infections by degrading foreign RNAs (sort of create antibodies, sense of immunity).
Double stranded RNas are normally part ,of viral origin or come form transposons (mobile elements who move from one part of genome to another, interfering with normal genome activity)
Process of degrading foreign RNAs:
1. in RNAi (RNA interference), Foreign double stranded RNA cleaved in to small fragments by DICER.
2. RISC will recognize double-stranded siRNA and use one single strand of siRNA (discard other strand) to search for complementary RNA
3. Once RISC find match with single stranded siRNA it has to compl. single-stranded foreign RNA, the foreign RNA will be degraded (infected cell turns foreign RNA against itself)
similar to miRNA, RISC will degrade foreign RNA with compl. sequences.
117
What is another function of siRNAs? How does this action occur?
siRNAs can also trigger transcription silencing.
artificial RNAI (RNA interference) is used to downregulate (SILENCE) specific genes in cultured cells and model organisms.
Process:
1. double-strands of siRNAS will be packaged together into protein called RITS Complex (RNA induced transcriptional silencing)
2. RITS Complex, will take single stranded form of siRNA and search for complementary RNA.
3. once found match, this will trigger modification of histones- as Histones will be METHYLATED, HETEROCHROMATIN formed, which will signal TRANSCRIPTIONAL REPRESSION.
this process can selectively shut off synthesis of foreign particles
118
What is the function and importance of long non-coding RNAs?
Long non-coding RNAs silence genes by similar mechanisms to miRNA, siRNA.
17,000 base-long non coding XIST RNA silences X Chromosome, by recruiting proteins that further condense one of the two x chromosomes.
many long non-coding RNAs are ANTISENSE RNA that are complementary to normal transcripts.
Through base pairing, they can form scaffolds that bring proteins together for same function.
