DNA & RNA, Transcription & Translation Flashcards
What does the enzyme RNA Polymerase I make?
rRNA (18S, 28S, 5.8S)
What does the enzyme RNA Polymerase II make?
mRNA, snRNA, miRNA, & IncRNA
What does the enzyme RNA Polymerase III make?
tRNA, 5S RNA, U6snRNA, & 7SK RNA
DNA and RNA grow in which direction?
5’ to 3’
Which bases are pyrimidines?
Cytosine & Thymine
Which bases are purines?
Adenine & Guanine
What is alpha-amanitin?
An inhibitor of eukaryotic RNA polymerase II
What is rifampicin?
An inhibitor of RNA polymerase; blocks RNA exit channel
What are the basic components of an nucleotide?
A pentose sugar, a purine or pyrimidine nitrogenous base attached to the 1’ carbon, and 1, 2, or 3 phosphates attached to the 5’ carbon. A DNA nucleotide has an OH group on the 3’ carbon of the pentose sugar. RNA has OH groups on both the 2’ and 3’ carbons.
Order from most soluble to least soluble: purines, pyrimidines, bases, nucleosides, nucleotides,
Pyrimidines > purines > nucleotides > nucleosides > bases
What is the central dogma of biological information transfer?
DNA replicates to make more DNA. DNA undergoes transcription to make RNA. RNA undergoes translation to make proteins.
Describe Gout and Lesch-Nyhan
Gout and Lesch-Nyhan are a failure to dissolve purines and purine byproducts in tissues. Purines are least soluble of all building blocks.
Identify the chemistry in the phosphodiester linkage of DNA and RNA polynucleotide strands.
Phosphate group on the 5’ carbon binds with the OH on the 3’ carbon
Describe the important experiments that helped to establish DNA as the genetic material
Avery, McCloud, and McCarty showed, using virulent and non-virulent bacteria, that DNA is the transforming material → aka, the genetic material.
Chargaff showed that % purines = % pyrimidines and that %G=%C, and %A = %T.
Rosalind Franklin and Maurice Wilkins used x-ray diffraction to show helical structure and 3.4 nm repeat.
Watson and Crick put together the double helix structure.
What are some nucleotides that are not in DNA or RNA?
cAMP, cGMP, NAD, Coenzyme A
What is the difference between a nucleoside and a nucleotide?
A nucleoside is a the pentose sugar plus a base. Adding phosphate groups makes it a nucleotide.
What is the complementary RNA strand of this DNA strand: 5’ AGCCTGTAGC 3’
5’ GCUACAGGCU 3’
How many Hydrogen bonds does one G-C bond make? One A-T bond?
A G-C bond makes three H bonds. An A-T bond makes two H bonds.
What are four conditions that will increase the melting temperature (stability) of DNA?
High salt concentration, high G-C concentration, longer strand, neutral pH.
What kinds of DNA are circular versus linear?
Mitochondrial and bacterial DNA
Where does methylation of DNA most commonly occur?
Methylation of Cytosine attached to 5’ carbon when this cytosine is adjacent to and just upstream of a guanine.
What is deamination of DNA and what are its consequences?
Deamination is the removal of an amino group and replacement of an Oxygen usually at a base. 5-Methylcytosine can be deaminated to become Thymine. There is now a 50/50 chance of making an mutagenic “correction.” Correction mechanism does not know whether C or T is the mutated base.
What is depurination and what are its consequences?
Purine bases are subject to attack by water, resulting in depurination: a break between the sugar and base.
What is UV Cross-linking and what are its consequences?
UV waves can cause two adjacent thymines to cross-link with one another.
What is oxidative damage and what are its consequences?
Hydroxyl radicals are generated by ATP production can cause OH groups to be attached to bases. Can cause downstream issues of DNA replication or transcription.
What are three classes of RNA in human cells and what are examples of them?
- Structural RNA ( rRNA, tRNA, snRNA small nuclear, and snoRNA small nucleolar)
- Regulatory RNA (control gene expression) = miRNA, siRNA (small interfering)
- Information-containing RNA = mRNA
What do topoisomerases do?
Relax supercoils in DNA
How does puromycin work?
Puromycin is a nucleotide analogue that mimics the tRNA acceptor region, allowing peptide transfer and termination. Puromycin will bind the ribosome and react with growing peptide chain to terminate translation (example of mimicry in antibiotics).
How can DNA replication be bidirectional if both strands must grow in the 5’ to 3’ direction?
There is a leading strand that grows continuously and a lagging strand that grows in Okazaki fragments.
What is the end-replication problem?
There is a progressive shortening of chromosomal ends during rounds of DNA replication. The leading strand can be synthesized to the very end but the lagging strand cannot be.
What do origin binding proteins do during DNA replication?
They recognized initiation sites on the DNA which are usually A-T rich. There are many many initiation sites on one DNA strand.
What do helicases do during DNA replication?
Unwind the double-stranded DNA
What do single-strand binding proteins do during DNA replication?
They bind to the single-stranded DNA to keep them from winding together again
What does primase do during DNA replication?
Creates an RNA primer for the replication of DNA
What does DNA Polymerase I do during DNA replication?
Removes RNA primer and replaces it with DNA. Is “distributive,” meaning it can come off easily and it does not use a sliding clamp.
What does DNA Polymerase III do during DNA replication?
Moves with a sliding clamp to make complementary new DNA strand.
What does DNA ligase do during DNA replication?
Ligates DNA fragments.
What does the sliding clamp do during DNA replication?
Allows DNA Polymerase III to stay clamped onto strand and slides in the 5’ to 3’ direction of new strand.
What does gyrase do during DNA replication?
Gyrase is a topoisomerase inhibited by quinolones. Found mostly in prokaryotic cells.
What does telomerase do during DNA replication?
Telomerase is a reverse transcriptase that elongates telomeres by using RNA templates to add DNA segments to ends of telomeres. This prevents the end-replication problem.
What does reverse transcriptase do during DNA replication?
Makes DNA from RNA template.
Describe how DNA polymerase creates the phosphodiester bond during addition of deoxyriboncleotides (dNTPs).
3’OH of newly added base attacks the alpha phosphate on 5’C of the incoming base, cleaving off a disphosphate group which provides the energy to create the phosphodiester bond
What is a point mutation? Insertion? Deletion?
Point Mutation is substitution of one base for another. Insertion is the addition of 1+ nucleotides in a sequence. Deletion is the deletion of 1+ nucleotides in a sequence.
What is deamination and which nitrogenous base does it affect?
Deamination is the removal of an amine group. The amine group of cytosine is removed, resulting in a uracil. A C-G pair is mutated to a U-A pair.
What are the consequences of oxidative damage to DNA?
Can block DNA replication or change a GC pair to a TA pair
What is alkylation and what are its consequences?
Alkylation is the addition of an alkyl group to guanine, turning it into methylguanine, which reacts with thymine.
What is DNA cross-linking? (Intra- and inter-strand)
When two nucleotide residues covalently connect (when they’re not supposed to). Intra- implies within the same strand; inter- implies with opposite strand.
What repairs a single-stranded break in DNA?
Ligase
What repairs UV-caused damage in DNA?
Photolyase
What repairs base alkylation damage in DNA?
O6-meG methyltransferase (MGMT
What is the difference between base excision repair and nucleotide excision repair?
Base excision repair repairs damages that do not distort DNA. NER repairs damages that do distort DNA.
What are the four steps of Base Excision repair?
Step 1. Modified base is recognized by a specific DNA glycosylase, which hydrolyzes the N-glycosidic bond, yielding an AP site.
Step 2. An AP site-specific endonuclease (APE1) cleaves the sugar-phosphate backbone 5’ to the AP site.
Step 3. Another endonuclease cuts 3’ to the AP site,
removing the deoxyribose phosphate.
Step 4. The resulting gap is filled by DNA polymerase, and the nick sealed by DNA ligase.
What are the four steps of Nucleotide Excision Repair?
- Recognition and binding of the damaged
site by a multi-protein complex (two
different ways depending on local
transcription activity). - Local unwinding of the DNA duplex by
helicases (parts of the TFIIH protein
complex) to form a bubble of ~25 bases. - Double incision of the damaged strand
By two endonucleases and removal of a
~30 base oligonucleotide containing the
lesion. - Filling in the gap by a DNA polymerase.
- Rejoining the two ends by a DNA ligase.
What are the two ways Nucleotide Excision Repair recognizes damage on DNA?
Global Genome NER & Transcription-coupled NER
Describe Xeroderma Pigmentosum
A defect in Global Genome NER. Symptoms include sun hypersensitivity, skin neoplasms, and cognitive degeneration later in life.
Describe Cockayne Syndrome
A defect in Transcription-Coupled NER. Symptome include sun hypersensitivity, progeria, impaired development, neurological degeneration.
Mismatch Repair (MMR) is initiated by which two protein complexes?
MutS and MutL Homologs
How does Mismatch Repair know which is the daughter strand and which is parent strand? (in humans)
Lagging strand daughter cell has many nicks that MutS H recognizes. Leading strand also has some, although fewer, nicks.
What causes Lynch syndrome?
Mutations in MMR genes.
What is damage/tolerance bypass (trans-lesion synthesis)?
When cells have too much DNA-repair for error-proof repair mechanisms, the cells employs “sloppy” DNA polymerases that do not have 3’-5’ exonuclease activity to replicate the DNA anyway.
What is the difference between Nonhomologous end-joining and Homologous recombination when repair double-stranded DNA breaks.
Nonhomologous end-joining simply rejoins ends, while Homologous recombination is more accurate and uses a homologous DNA strand to rejoin ends.
What does a single strand break activate in order to repair the break?
PARP: Poly(ADP-ribose) polymerase; adds poly(ADP-ribose) chains to proteins
Which subunits make up bacterial ribosomes? Eukaryotic?
50S + 30S = 70S (prokaryotic). 60S + 40S = 80S (eukaryotic)
What are the three sites in a ribosome?
PAE: Aminoacyl site, peptidyl site, exit site
What is the first step in the initiation step of bacterial translation?
Initiation factors IF1 and IF3 bind the 30S subunit to the mRNA using the Shine-Dalgaro sequence.
Is initiation of translation the same in prokaryotes and eukaryotes? Is elongation?
Initiation in eukaryotes is much more complex so as to allow more regulation. Elongation is virtually identical, except some factors have different names.
Where on the large ribosome is the peptide bond formed during translation?
In the Peptidyl Transferase Center (PTC)
How many high-energy bonds does it “cost” to make one peptide bond?
Four
What is a missense mutation in translation?
A change in a base that results in a change of amino acid.
What is a silent mutation in translation?
A change in a base that results in no change of amino acid, due to redundancy
What is a frame-shift mutation in translation?
If a nucleotide is added or deleted, it shifts the entire codon sequence so as to create completely different coding sequence
What is a nonsense mutation in translation?
Mutation resulting in a premature stop codon
What is a sense mutation in translation?
Mutation resulting in the absence of a stop codon
Describe the disease “Hemoglobin Wayne”
3’ terminal frameshift mutation - associated with chronic anemia
Describe the disease “Hemoglobin Constant Spring”
A normal UAA stop is mutation to a CAA Gln - associated with chronic anemia
What is transferrin?
A molecule that binds iron during iron metabolism
What is Transferrin Receptor (TFR)?
Transports transferrin/Fe into cell
What is Iron Response Element (IRE)?
RNA stem-loop structure found in mRNAs that can bind to IRPs (Iron Response Binding Porteins)
What are Iron Response Binding Proteins 1 & 2 (IRE-BPs)?
Bind Fe & regulate expression of ferritin and Transferrin Receptor
What is Ferritin?
Sequesters excess Fe (Iron) and places it into a non-toxic storage form
What does mTOR regulate? (in general)
Translation
What does eIF-2alpha do once it is phosphorylated and inactivated, in terms of translation?
It inhibits ternary complex formation - no initiator tRNA is delivered to ribosome to initiation translation
What do the antibiotics streptomycin, erythromycin, tetracycline, and chloramphenicol target?
Ribosome
What does an interferon do?
When a virus affects a cell, the cell releases interferon, which warns other cells of viral infection and initiates stress response pathways.
Are introns or exons of pre-mRNA the coding sequences?
Exons
To initiate transcription, polymerase binds to DNA, then creates a bubble in DNA, then catalyzes a phosphodiester linkage of two initial ________.
rNTPs
How does alpha-amanitin from the death cap mushroom block transcription?
It is a non-competitive inhibitor of RNA Pol II. It binds the bridge helix and blocks RNA chain elongation by preventing translocation.
How does the antibiotic Rifampicin work?
Binds bacterial RNA polymerase and blocks the RNA exit channel
TFIIH is an initiation co-factor for which enzyme?
RNA Pol II
What are three syndromes associated with mutations in TFIIH?
Xeroderma pigmentosum, Cockayne’s syndrome, & Trichothiodystrophy
What replaces the 5’ phosphates of pre-mRNA?
7-methyl-guanosine cap
The 5’ mRNA cap is added by which enzymatic steps?
- Triphosphatase
- Guanylyltransferase
- Guanine 7 methyl transferase
What recognizes the 5’ splice site of pre-mRNA?
U1 snRNA
What recognizes the branch point of pre-mRNA?
U2 snRNA
What are the components of the spliceosome?
It is a large ribonucleoprotein complex composed of pre-mRNA, 100+ proteins, and 5 small nuclear RNAs
What are the two transesterification steps involved in splicing?
1: Attack by the 2’ OH of the branch point A
2. Attack by the 3’ OH of exon 1
What does alternative splicing allow?
Allow many different proteins to be encoded by a single gene
What are the two enzymatic steps of 3’ mRNA polyA adenylation?
- Cleavage
2. Polyadenylation
What is the base sequence of the polyA site on pre-mRNA?
AAUAAA
In the cytoplasm, what is the 5’ cap replaced by?
Translation initiation factor eIF4E
What sequence marks the start of the 5’ end of an intron? The 3’ end
5’ GU 3’ AG
What recognizes the 3’ splice site?
U2AF (association factor)
