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Biochemistry Block Two
Terms in this set (176)
Which are purines?
Adenine and Guanine
Which are the pyrimidines?
cytosine, thymine, uracil (in rna)
How are unusual (modified) bases produced?
Occurs by post-transcription modifications
What is the unusual (modified) base commonly found in DNA?
What is the unusual (modified) base commonly found in RNA?
What is the common step in both purine and pyrimidine synthesis?
What is the activator and inhibitor of PRPP?
Inhibitor- end product (nucleotides)
What is PRPP synthesized from?
ATP and ribose 5-phosphate
What does AMP synthesis require?
GTP and asparate
What does GMP synthesis require?
ATP and glutamine
Both AMP and GMP comes from
What enzyme is needed to go from GDP/ADP to GTP/ATP?
What enzyme is needed to go from AMP to ADP?
What enzyme is needed to from GMP to GDP?
What are the two enzymes needed in the purine salvage pathway?
The purine salvage pathway is what?
Which purine pathway is the most important in the brain?
Purine salvage pathway
What is Lesch-Nyhan syndrome?
Defective purine salvage owing to absence of HGPRT.
Results in excess uric acid production and de novo purine synthesis
About ___ of purines synthesized via de novo are then used in purine salvage pathway. Other ___ goes straight to uric acid
of purine synthesized via de novo are in purine salvage pathway. Other
goes straight to uric acid
When does synthesize of deoxyribonucleotides occur?
The S-phase of the cell cycle
What enzyme is responsible for making deoxyribonucleotides?
What is the structure of ribonucleotide reductase?
A dimer with 2 nonidentical subunits: alpha and beta
What is the only enzyme used to create ALL the deoxynucleotides?
What is the protein (co-enzyme) that is associated with ribonucleotide reductase and sorta helps it do it's job?
What are the two allosteric sites on ribonucleotide reductase?
Specificity and Activity sites (2 each, 4 total)
What has to be bound to activate ribonucleotide reductase?
ATP on the active site
What inhibits ribonucleotide reductase?
dATP bound to the active site
The binding of ATP to the active site of ribonucleotide reductase ensures that...
you produce a
amount of deoxynucleotides for DNA synthesis
What is the end product of purine nucleotide degradation?
What is gout?
Too much uric acid in blood causes urate crystals to form in the joints
What is arthrocentesis?
surgical puncture to remove fluid from the joint space (popular for helping gout)
What is the treatment for acute gout?
- steroidal drugs
- nonsteroidal drugs
What is the treatment for chronic gout?
-Febuxostat (for people who can't take allopurinol)
When you decrease THF (tetrahydrofolate) you have
the inhibition of purine synthesis, dTMP, and DNA synthesis that
slows cell growth
What does methotrexate do?
Binds to and inhibits the production of THF (tetrahydrofolate) that then results in the inhibition of purine nucleotide synthesis and ultimately
slows cell growth
DNA polymerase III is the
Principle replication polymerase
DNA polymerase I primary function is
What are DNA polymerase II, IV, and V involved in?
In DNA repair
What strands make Okazaki fragments?
the lagging strand
What does DNA polymerase require? What does it contain?
A primer that contains a 3' hydroxyl group that begin the first DNA polymerase-catalyzed reaction
What does primase synthesize?
A short RNA strand (about 5 nucleotides) complementary to DNA
What does DNA polymerase I do in relation with the primers?
It removes the primer and replaces RNA with DNA using it's 5' to 3' exonuclease activity
What makes DNA polymerase so highly processive?
The sliding clamp- it causes the polymerase to not dissociate from the template strand
What does highly processive mean?
Once you begin catalysis it rarely releases the DNA substrate until it's finished.
What makes up the DNA polymerase III holoenzyme?
Has 2 core subunits linked together by the "clamp loader" complex with a helicase attached. The 2 beta subunits come together to form the sliding clamp
What activity does ALL polymerase have?
3' to 5' exonuclease activity (proofreading)
What are exonuclease? How do they work?
enzymes that cleave nucleotides one at a time from the end via hydrolyzing reaction that breaks the phosphodiester bonds
What makes DNA polymerase I special?
It has the 3' to 5' exonuclease activity AND
5' to 3' exonuclease activity
that makes it the primary 'repairman'
What is nick translation and who uses it?
DNA polymerase I uses it to degrade the strand ahead of the enzyme and then synthesizes new strand behind it
What are telomeres?
'Caps' on the end of chromosomes (form special loop structures to keep DNA ends from unraveling)
What does telomerases do?
Add the sequences to form the telomere on the end of a chromosome. A type of reverse transcriptase
What activity is characteristic of cancer cells?
High telomerase activity
Overview of DNA repair mechanisms
-Detection of the lesion
-Removal of damaged DNA (by glycoylases and nucleases)
-Repair (by DNA polymerase and DNA ligase)
-Regulated by proteins (protein kinase)
How does nucleotide excision repair work?
Damaged pairs are cut out and DNA polymerase fills in the gap and ligase seals it to phosphate backbone.
in both eukaryotes and prokaryotes
In repairing DNA who is responsible for removing the damaged section, filling in the gap, and sealing the strand?
Endonucleases remove damaged sections, DNA polymerase fills in the gaps, and DNA ligase seals the strand
What Mut proteins are in methyl-directed mismatch repair in E. Coli?
How does methyl-directed mismatch repair work?
-Mut S first forms a dimer on the daughter strand
-Mut L finds Mut S and mediates the action of Mut S
-Mut H is then activated and nicks the daughter strand at the methylated site
In methyl-directed mismatch repair, how do we know which strand to fix?
The parent strand is methylated, so the unmethylated daughter strand is the one to be fixed
How does base excision repair work?
Glycolases have the ability to flip out the nucleotides in order to detect a single base error- which it then hydrolyzes the bond to remove it (now an AP endonuclease site)
What is DNA double strand break repair?
They commonly repair genomic rearrangements (very common in cancers) via a "cut-and- paste" mechanism
What are the two types of DNA double strand break repair?
1. Homologous recombination: S and G2 phase
2. Non-homologous end-joining: G1 phase
What are transposable elements?
"jumping genes"- a DNA sequence that moves from one location on genome to another
How much of the human genome is made up of transposable elements?
What are the types of TEs?
1. Retrotransposons (require reverse transcriptase)
2. DNA transposons
What is rRNA? What does it do?
Ribosomal RNA- function is to bind to mRNA and tRNA to ensure that the codon sequence of the mRNA is translated accurately into amino acid sequence in proteins
What is the important species of rRNA?
16s (in prokaryotes)
What is the order of what RNA from most common to least?
rRNA> tRNA> mRNA
What is the catalytic role of rRNA?
form peptide bonds in protein synthesis
What is tRNA and what is it's function?
transfer RNA- helps decode a mRNA sequence into a protein
What do tRNA usually contain?
Modified (unusual) bases
At what site do the amino acids attach to in tRNA?
the 3' end has CCA sequence
What does the anticodon on tRNA do?
recognize genetic code on mRNA
What is mRNA and what is its function?
Messenger RNA- serves as direct carrier of genetic information from genes to ribosomes
What does polycistronic mean?
mRNA carries code for more than one gene (in prokaryotes)
What does monocistronic mean?
mRNA carries code for one gene (eukaryotes)
In eukaryotes what protects mRNA?
5' cap and the 3-poly-A tail on the 3' end
How many RNA polymerases do prokaryotes have?
One- it does it all!
What does RNA polymerase do?
-synthesizes RNA copy via 5' to 3'
Why don't RNA polymerase need primers?
Because they start precisely where the genes start thanks to promoter sequences
What is RNA polymerase holoenzyme?
5 core enzymes: 2 alpha, 2 beta, omega
bound to a sigma factor
What does the sigma factor on RNA polymerase holoenzyme do?
Recognizes the promoter region
What are the steps in transcription elongation?
1. RNA pol continues to unwind DNA
2. RNA pol uses NTPs as substrates
3. Transcript synthesis begins when you produce a strand >10 nt is produced (shorter ones are just discarded)
4. Sigma factor is released (RNA pol is no longer a holoenzyme)
5. Core enzymes of RNA pol clears the promoter and moves along the template
6. A RNA-DNA hybrid is formed
What is rho-independent termination?
-RNA is transcribed from GC-rich region can fold back on itself (due to H+ bonding) and forms hairpin structure
- Now we have weak A-U basepairing that dislodges the RNA polymerase from the DNA template
-This happens while DNA strand starts to reanneal behind the stalled RNA polymerase
-Causes primary mRNA and signals termination of RNA synthesis
Which termination is intrinisic?
What is rho-dependent termination?
-Rho unwinds RNA transcript from the DNA using its helicase activity
- It chases behind DNA polymerase and eventually catches up and causes it to release transcript
What is relaxed chromatin?
What is tight chromatin called?
What is it called when we go from heterochromatin to euchromatin? How?
Chromatin remodeling via covalent modifications of histones
What does HAT (histone acetyltransferase) do?
Removes the + on lysine within the histone by acetylation that causes DNA affinity to the histone to decrease. Turns it into euchromatin
What does HDAC (histone deacetylases) do?
Restores the + charge on lysine so DNA affinity to the histone increases and we form heterochromatin
What are enhancers and what do they do?
Special DNA sequences that increase the rate of initiation of transcription by RNA pol II
Where can enhancers be?
Anywhere! Upstream, downstream, close, far away, same strand, other strand
How do enhancers work?
They bend DNA to allow STF to interact with TF-promotor-RNA pol II complex and stimulates transcription
Once you splice something you make
a completely different protein
What percentage of pre-mRNA can be spliced?
a diverse set of proteins to be produced from a limited set of genes
What is charged tRNA?
tRNA covalently attached to an amino acid
What is uncharged tRNA?
tRNA without an amino acid
What are the two important structures to know on tRNA?
Anticodon and CCA on 3' end
What does aminoacyl-tRNA synthetase do?
Charges tRNA by attaching the amino acid
Each amino acid has its own aminoacyl-tRNA synthetase which allows
high fidelity translation
What is the mechanism in which ribosomes recognize nt sequence to initiate translation in prokaryotes?
How does the shine- dalgarno sequence work in prokaryotes?
A special sequence that is near AUG and causes the 16s rRNA binds to the 30s subunit of mRNA so that translation starts
What is the mechanism in which ribosomes recognize nt sequence to initiate translation in eukaryotes?
How does the 5'-cap work in eukaryotes?
eLF-4 proteins with some ATP facilitate the binding of rRNA large subunit near the 5'-cap on mRNA
What is tRNAi?
A special tRNA that recognizes AUG
How does tRNAi recognize the start codon?
-IF-2-GTP in prokaryotes
-eLF-2-GTP in eukarytoes
Where does tRNAi go?
Directly to the P-site
What does tRNAi carry in both prokaryotes and eukaryotes?
Prokaryotes carry fMet
Eukaryotes carry Met
Steps to elongation
1. Charged tRNA delivered to A-site
2. Ribozyme catalyzes peptide bond between amino acid in P-site and amino acid in A-site
the peptide on tRNA P site move to A site on tRNA
P site is now uncharged so moves to E site. A site peptidyl-tRNA moved to P site
5. A site is ready to accept new charged tRNA
6. Repeats until hits stop codon
When does termination occur?
When one of the three stop codons (UAA, UGA, UAG) moves into the A site on tRNA. (recognized by release factors)
Gene expression most commonly regulated at transcription, why?
Translation takes a lot of energy so stops it early to save energy
What is one way to regulate translation in eukaryotes?
eIF-2 brings tRNAi to the P-site. BUT if you phosphorylate eIF-2 then you can not bring tRNAi to the P-site
What is the way to regulate translation in BOTH eukaryotes and prokaryotes?
RISC (RNA induced silencing complex) binds to mRNA and blocks ribosome assembly of clearance
How do proteins made in the cytoplasm go to where they are needed to function?
Proteins have an amino acid sequence that direct them to their final locations
How does protein targeting work? (secreted protein: translational targeting)
1. N terminal sequence on growing peptide is recognized by
(signal recognition particle)
2. SRP binds the ribosome and halts elongation
3. SRP delivers this half elongated peptide complex to the RER membrane
4. SRP receptor undergoes translocon to allow complex in to RER
5. Translation continues on
6. Protein moves through RER then Golgi and leaves cell
What are structural genes?
Codes for any RNA or protein product other than a regulatory protein
What are operons?
coordinately regulated gene clusters
What makes up an operon?
Promoter and structural genes
What happens to lactose operon when only glucose present?
Lac operon is repressed (turned off)
What happens to lactose operon when only lactose present?
Max production of the 3 proteins (Beta glatatosidase, permease, tradacetylase)
What happens to lactose operon when both lactose and glucose present?
Lac operon is uninduced so very low expression of the three proteins (because your body will use glucose first but if lactose there we wont completely waste it)
What is special about the tryotophan operon in E.Coli?
It can be regulated via attenuation when repression is incomplete (backup system)
Why can attenuation only occur in prokaryotes?
Because only in prokaryotes can mRNA translation begin while mRNA synthesis is still happening
What happens when trp levels high?
Operon is repressed
What happens when trp levels low?
What happens when trp level are high
transcription is still being initiated?
It's called escaped repression and attenuation kicks on to full repress the trp operon
Why are operons only in prokayotes?
In eukaryotes each gene is makes an individual mRNA and each gene has it own promoter
What are the two ways mRNA is stabilized?
The longer the mRNA stays in the cytosol the more ____ it makes
the longer the mRNA stays in the cytosol the more proteins it makes
What happens when iron concentration is low?
-Iron regulatory proteins bind to the 3'-Iron responsive element
-Stabilizes the mRNA for tranferrin receptor synthesis
-increase transferrin receptor synthesis to help bring in more iron into the cell
What happens when iron concentration is high?
-iron regulatory proteins dissociated from 3'-iron responsive element
-mRNA for tranferrin receptor is unstable and degraded
-Lower tranferrin receptor synthesis
What can microRNA do?
Degrade/ repress translation (affects mRNA stability)
How does RNA interference (RNAi) work?
1. miRNA transcribed from DNA to pri-miRNA
2. Drosha processes pri-mRNA into pre-miRNA
3. Pre-miRNA now travels to cytoplasm
4. Dicer processes pre-miRNA into dsRNA
5. One miRNA strand (guide) associates with RISC
6. miRNA-RISC binds with complementary sequence of target mRNA
7. Target mRNA is translationally repressed or degraded
What are indels and what do they do?
Insertions and deletions. Both are frameshift mutations
What can happen when you change the reading frame?
- Product will be completely different amino acid sequence
- Truncated product due to premature introduction of termination/ stop codon
How do you know its autosomal dominant?
-equal number of males and females affected
How do you know its autosomal recessive?
-equal number of males and females affected
How do you know its X-linked dominant?
-A father has= every daughter will have
How do you know its X-linked recessive?
What is the defect in hypoplastic (type of amelogenesis imperfecta)?
enamel matrix formation
What is the defect in hypocalcified (type of amelogenesis imperfecta)?
What is the defect in hypomaturation (type of amelogenesis imperfecta)?
What mutation is the most common in amelogenesis imperfecta?
Amelogenesis imperfecta caused by FAM83H mutation is inherited via?
Amelogenesis imperfecta caused by MMP20 mutation is inherited via?
Amelogenesis imperfecta caused by ENAM mutation is inherited via?
Amelogenesis imperfecta caused by AMELX mutation is inherited via?
X-linked. Males experience more severe abnormalities
Where are the two main cell cycle checkpoints at?
First one: G1/S
Second one: G2/M
Interphase: G1, S, G2
M phase: PMAT
Chromosomes become visable, nucleolus dissolves, spindle forms
Disassembly of nuclear envelope
Chromosomes line up in the middle of the cell
Phase of mitosis in which the chromosomes separate and move to opposite ends of the cell
Microtubules disassemble, nuclear envelope reforms, chromatids decondense
What are the cell cycle regulators?
Cyclins and cyclin-dependent kinases (CDK)
G1 regulators are
S phase regulators
E, A cyclins
Mitotic phase regulators
B, A cyclins
What does retinoblastoma (RB) protein do?
Halts the cell in G1, used as a tumor suppressor
Not programmed cell death, the cell blows up, takes several days
Programed cell death, Cyctochrome C leaves mitochondria, cell shrinks, membrane blebs, completed
What enzymes make
What enzymes make
Cohesive ends are
Blunt ends are
Even-length ends from both single strands, cuts down the middle
What do blunt ends need to join back together?
Bacteriophage T4 ligase
What are plasmid vectors?
small, circular molecules of double-stranded DNA derived that occur naturally in bacterial cells
What is transformation?
process of introducing foreign DNA into bacteria or yeast cells
What is hybrid plasmid formation?
1. Cleave wanted gene
2. Amplify DNA (makes sticky ends)
3. DNA ligase joins DNA and plasmid
4. Recombinant DNA introduced into host by transformation
5. Cell reproduce that makes more of the wanted gene (that you wanted to study)
Polymerase Chain Reaction Steps and temperatures
1st denature @ 90-100
2nd anneal @ 30-65
3rd elongate @ 60-70
What sample is used in southern blot?
What sample is used in northern blot?
What sample is use in western blot?
What sample is used in microarray?
What sample is used in ELISA?
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