Biochem Ch 1-3 Flashcards
chemo drugs that act in S phase
- methotrexate, 5-flurouracil, hydroxyurea
- interfere with thiamine (only in DNA) synthesis
chemo drugs that act in G2 phase
bleomycin
chemo drugs that act in M phase
paclitaxel, vincristine, vinblastine
non cell-cycle specific chemo drugs
cyclophosphamide, cisplatin
Nucleoside
base + 5C sugar
Pyrimidines
- Cytosine, uracil, thymine
- “CUT the Py”
- if you deaminate C, you get U
- Uracil is only in RNA
- difference between U and T = T has a methyl group
- Thymine is only in DNA
- Cytosine is in both DNA/RNA
- structure is 1 ring
- “More complex name, more simple structure”
Nucleoside of Adenine and Guanine
Adenosine (deoxyadenosine), Guanosine (deoxyguanosine)
Nucleotide
base + 5C sugar + phosphate
Nucleoside of cytosine
cytidine (deoxycytidine)
nucleoside of uracil
uridine (deoxyuridine)
nucleoside of thymine
deoxythymidine (BC only in DNA)
MOA for daunorubicin and doxorubicin
- Anti tumor drugs used to treat leukemias
- intercalating between the bases of DNA, thereby interfering with the activity of topoisomerase II and preventing proper replication of the DNA
Purines
- Adenine and Guanine
- if you deaminate A you get G
- Purines have 2 ringed structures
- “PURe As Gold”
MOA of cisplatin and use
- antitumor drug used to treat bladder and lung tumors
- bind tingtly to the DNA, causing structural distortion and malfunction
negative vs positive supercoiling
negative superoiling = DNA is wound MORE LOOSELY than watson-crick DNA
positive supercoiling = DNA is wound MORE TIGHTLY than watson-crick DNA
How to topoisomerases work? and example
They change the amount of supercoiling in DNA molecules by making transient breaks in DNA strands by alternately breaking and resealing the sugar-phosphate backbone.
ie E. Coli DNA gyrase (DNA Topoisomerase II) can introduce negative supercoiling in DNA
nucleosome
DNA wrapped 2x around 8 histones: (2 H2A, 2 H2B, 2 H3, 2 H4 = a histone octamer)
H1 = associated w the linker DNA found between the nucleosomes to help package them into a solenoid-like structure (a thick 30 nm fiber)
what amino acids predominate in histones?
lysine and arginine. both have a + charge and confer a positive charge on the proteins
Why does DNA have a negative charge?
because between every 5C sugar there is a phosphate with a negative charge
euchromatin
- light staining
- transcripturally active genes
- apoptois
- more opened and available for gene expression
- ie insulin gene in pancreas
heterochromatin
- dark staining
- transcripturally inactive genes
- highest order packaging
- condensed and associated with areas of chromosomes that are NOT expressed
- ie insulin gene in muscle
- MITOTIC DNA = MOST CONDENSED DNA to allow separation of the sister chromatids= no gene expression.
anything ending in “-in”
protein!
Double stranded DNA associates together via
hydrogen bonding
nucleotide
phosphate group is attached to the 5’ C of a nucleoside
characteristic features of eukaryotic cell death (in nucleus) by apoptosis
endonuclease activation and chromatin fragmentation
circular DNA double helices
- bacterial chromosome
- bacterial plasmid
- mitochondrial chromosome
most viral genomes are
SSDNA
- an example of an exception = parvovirus
mitochondrial DNA is found in the
cytoplasm (like in bacteria)
RNA is made in the (location)
nucleus and is moved to the cytoplasm
examples of cells in G0
cells that don’t replicate
- muscle, liver, nerve cells
- these cells still undergo transcription, because gene EXPRESSION does occur
When is RNA made?
Throughout interphase (G1-S-G2) - DNA is only made in S (active replication in nucleus)
Signal to go into S
growth factor
polymerases do what
synthesize Nucleic acids by forming phosphodiester bonds (a type of covalent bond)
- DNA polymerase proofreads DNA as it makes it
- dNTP substrates are added and PPi (inorganic phosphate) are then removed off the 3’ end
- bc dNMP are in chain but in the nucleoplasm they exist as dNTP
nucleases do what?
hydrolyze phosphodiester bonds
exonucleases do what?
remove nucleotides from either the 5’ or 3’ end of a nucleic acid
endonucleases do what?
cut within the nucleic acid and release nucleic acid fragments
There are how many genes on how many chromosomes?
20-25K genes on 46 chromosomes
AA switched in sickle cell anemia
glutamate changed to a valine
how are nucleotides added
- when the 3’ hydroxyl group of the growing strand reacts with a nucleoside triphosphate, which is base-paired with the template strand.
- pyrophosphate (PPi, the last two phosphates) are released during this reaction
DIFFERENCES btw DNA and RNA synthesis: SUBSTRATES
- the substrates for DNA synthesis are dNTPs, whereas the substrates for RNA synthesis are NTPs
DIFFERENCES btw DNA and RNA synthesis: bases
- DNA has thiamine, RNA has uracil
DIFFERENCES btw DNA and RNA synthesis: primers
- DNA polymerases require a primer, whereas RNA polymerases do not
- DNA polymerases cannot initiate strand synthesis, whereas RNA polymerases can
- DNA polymerases require a RNA primer because they cannot bind to SSDNA
DIFFERENCES btw DNA and RNA synthesis: proofreading
DNA polymerasess have proofreading and can correct mistakes
- DNA polymerases have 3’ to 5’ exonuclease activity for proofreading
- RNA polymerases can’t. This isn’t a big deal because RNA is short-living
Helicase
breaks the hydrogen bonds holding the base pairs together. This allows the 2 parental strands of DNA to begin unwinding and forms 2 replication forks
SSB
single-stranded DNA Binding Protein:
- binds to the single-stranded portion of each DNA strand, preventing them from reassociating and protecting them from degradation by nucleases
primase
- synthesizes a short ( approx 10 nucleotides) RNA primer in the 5’ to 3’ direction. beginning at the origin on each parental strand
- the parental strand is used as template for this process
- RNA primers are required bc DNA polymerases can’t initiate synthesis of DNA
- DNA polymerases can only extend a strand from 3’ end of a pre-formed primer
how are okazaki fragments made
- each fragment is initiated by the synthesis of an RNA primer by primase
- then completed by the synthesis of DNA using DNA polymerase III
- each fragment is made in the 5’ to 3’ direction
How are RNA primers removed
- RNA primers are removed by RNAse H in eukaryotes
- and an uncharacterized DNA polymerase fills in the gap with DNA
- in prokaryotes DNA polymerase 1 both removes the primer (5’ exonuclease) and synthesizes new DNA, beginning at the 3’ end of the neighboring okazaki fragment.
DNA gyrase
- aka DNA topoisomerase II
- provides a swivel in front of each replication fork
- as helicase unwinds DNA at the replication forks, the DNA ahead of it becomes overwound and + supercoils form (aka DNA is wound MORE TIGHTLY than watson-crick DNA)
- DNA gyrase inserts negative supercoils (aka DNA is wound MORE LOOSELY than watson-crick DNA) by nicking both strands of DNA, passing the DNA strands THROUGH the nick, and then resealing both strands.
- this helps make DNA more stable
- it is not working in G0!
- inhibited by Nalidixic acid (how it kills bacteria)
DNA polymerases can only extend a strand from
the 3’ end of a preformed primer.
- DNA is made 5’ to 3’, which means the template is READ 3’ to 5’.
Quinolones function
- quinolone and fluoroquinolones:
- inhibit DNA gyrase (prokaryotic topoisomerase II) preventing DNA replication and transcription
- thus destabilizing DNA of bacterial cells!!
- these drugs are most active against aerobic gram-negaitve bacteria
- examples: levofloxacin, ciprofloxacin, moxifloxacin
- current uses include treatment of gonorrhea and upper and lower UTI in both sexes
- TEST QUESTION!
etoposide, teniposide
- inhibitors of eukaryotic topoisomerase II that are becoming useful anticancer agents
- affect rapidly dividing cells in GI, epithelial
function of telomerase, what is involved with telomerase (its structure/action)
- completes the replication of the telomere sequences at both ends of a eukaryotic chromosome
- it contains a short RNA template complementary to the DNA telomere sequence, as well as telomerase reverse transcriptase activity (hTRT)
- so it can replace telomere sequences that would otherwise be lost during replication
where do you find telomerase
present in embryonic cells, fetal cells, and certain adult stem cells
- NOT PRESENT IN ADULT SOMATIC CELLS
- inappropriately present in some cancer cells, leading to their inappropriate, unlimited replication
telomeres
- repetitive sequences at the ends of linear DNA molecules in eukaryotic chromosomes
- in each round of replication in most normal cells, the telomeres are shortened because DNA polymerase can’t complete synthesis of the 5’ end of each strand.
- this contributes to the aging of cells, because eventually the telomeres become so short that the chromosomes can’t function properly and cells die.
Function of AZT
- chemotherapeutic drug used to treat HIV
- once it enters cells, it can be converted to the triphosphate derivative and used as a substrate for the viral reverse transcriptase in synthesizing DNA from its RNA genome
- the replacement of an AZIDE instead of a normal hydroxyl group at the 3’ position of the deoxyribose prevents further replication by effectively causing CHAIN TERMINATION
- although it is a DNA polymerase, reverse transcriptase lacks proofreading activity!
Nalidixic acid MOA
kills bacteria by inhibiting DNA gyrase
synthesis of leading and lagging strands in prokaryotic cells
DNA polymerase III
removal of RNA primers in prokaryotic cells
DNA polymerase I (5’ to 3’ exonuclease)
- DO NOT CONFUSE WITH 3’ to 5’ EXONUCLEASE
- The 3’ to 5’ human type endonuclease is known to be essential for the proper processing of histone pre-mRNA
What replaces RNA with DNA
- in prokaryotic cells = DNA polymerase I.
- in eukaryotic cells = unknown
alpha carboxyl group on AA has a PK of?
- approx 2
- at physiologic pH of 7.4 the alpha carboxyl group on AA will be deprotonated (ionized) = COO-
the alpha amino group on AA has a PK of ?
- approx 9
- at physiologic pH of 7.4 the alpha amino group on AA will remain protonated = NH3+
synthesis of leading and lagging strands in eukaryotic cells
DNA polymerases alpha and delta
removal of RNA primers in eukaryotic cells
RNAse H (5’ to 3’ exonuclease)
DNA synthesis by reverse transcriptase in retroviruses can be inhibited by
- AZT
- ddC
- ddI
reverse transcriptase activity in eukaryotic cells
- associated with telomerase (hTRT)
- encoded by retrotransposons (residual viral genomes permanently maintained in human DNA) that play a role in amplifying certain repetitive sequences in DNA
SE of quinolones
- phototoxicity
- tendinopathy!
- tendon rupture
quinolone are contraindicated
pregnancy and children
- bc affects chondrogenesis
p53 gene
- encodes a protein that prevents a cell with damaged DNA from entering the S phase
- aka a Tumor Suppressor Gene
- p53 gene inactivation or deletion associated with Li-Fraumeni Syndrome (AD) and many solid tumors
ATM gene
- encodes a kinase essential for p53 activity
- ATM gene inactivated in ataxia telangiectasia, characterized by hypersensitivity to x-rays and predisposition to lymphomas
BRCA-1 gene associated with
breast, prostate and ovarian cancer
BRCA-2 gene
breast cancer
xeroderma pigmentosum mutation
- extreme UV sensitivity
- excessive freckling
- multiple skin cancers
- corneal ulcerations
- carcinomas and melanomas appear early in life and most patients die of cancer (patients should avoid exposure to any source of UV light)
- AR
- actinic keratosis = premalignant condition
- excision endonuclease (corrects thiamine dimers) deficient in xeroderma pigmentosum
- Presents in kids
- also may see slight stature and poor muscle tone
hereditary nonpolyposis colorectal cancer = HNPCC
- aka lynch syndrome
- mutation on one of two genes, hMSH2 or hMLH2 (one finds mismatched base and the other takes it out)
- mutation results in defective repair of DNA mismatches
- AD
- better prognosis than sporadic colon cancer
prokaryotic RNA polymerase is inhibited by
prokaryotic RNA polymerase is inhibited by rifampin
Eukaryotic DNA polymerase(s) that synthesize both the leading and lagging strands
DNA polymerase alpha and delta work together to synthesize the leading and lagging strands
Eukaryotic DNA polymerase(s) that replicates mitochondrial DNA
DNA polymerase gamma replicates mitochondrial DNA
Eukaryotic DNA polymerases beta and epsilon
- Eukaryotic DNA polymerases beta and epsilon are thought to participate primarily in DNA repair
- DNA polymerase epsilon may substitute for DNA polymerase delta in certain cases
The majority of cases of cystic fibrosis result from the…
- The majority of cases of cystic fibrosis result from the deletion of phenylalanine at position 508
- this interferes with proper protein folding and the post translational processing of oligosaccharide side chains
- abnormal chloride channel protein (CFTR) is degraded by the cytosolic proteasome complex rather than being translocation to the cell membrane
Summary: CF results from a misfolded protein
Gray baby syndrome
- a dangerous condition that occurs in newborns (especially premies) who’re given chloramphenicol (drug given to fight bacterial infections, including meningitis)
- if given to a newborn, this can trigger a potentially deadly reaction
- babies do not have sufficient UDP-glucuronyl transferase activity needed to allow excretion of this drug
- the drug builds up in the baby’s bloodstream and can lead to:
- blue lips, nail beds and skin (cyanosis)
- death
- low BP
what is the most common RNA?
rRNA
Li-Fraumeni Syndrome
- AD
- p53 gene inactivation or deletion
- rare disorder that greatly increases the risk of developing several types of cancer, particularly in children and young adults.
- cancers most often associated with Li-Fraumeni syndrome: breast, osteosarcoma, soft tissue sarcomas, brain tumors, leukemias, and adrenocortical carcinoma that affects the outer layer of the adrenal glands
ataxia telangiectasia
- characterized by hypersensitivity to x-rays and predisposition to lymphomas
- ATM gene inactivated in ataxia telangiectasia
- ATM gene encodes a kinase essential for p53 activity
Rb gene
- first Tumor Suppressor gene ever cloned
- negative regulator of the cell cycle through its ability to bind the transcription factor E2F and repress transcription of genes required for S phase!!!
most DNA repair occurs during the ___ phase of the eukaryotic cell cycle
most DNA repair occurs during the G1 phase of the eukaryotic cell cycle
Mismatch repair occurs during the
mismatch repair occurs during the G2 phase to correct replication errors
- a mutation on one of two genes, hMSH2 or hMLH2 (one finds base and one takes it out) initiates defective repair of DNA mismatches, resulting in a condition known as hereditary nonpolyposis colorectal cancer = HNPCC = lynch syndrome
- mismatched bases are repaired by DNA polymerase (replaces missing pieces of sequence: fills in gap by synthesizing DNA in the 5’ to 3’ direction, using the undamaged strand as a template) and DNA ligase (DNA ligase makes phosphodiester bond = type of covalent bond)
Thiamine dimers
- cause
- recognized/excised by
- repaired by
- when during the cell cycle does this repair occur?
- caused by UV radiation
- recognized and excised by excision endonuclease (deficient in xeroderma pigmentosum, which presents in kids)
- repaired by DNA polymerase (replaces missing pieces of sequence: fills in gap by synthesizing DNA in the 5’ to 3’ direction, using the undamaged strand as a template) and DNA ligase (DNA ligase makes phosphodiester bond = type of covalent bond)
- this repair occurs during G1
cytosine deamination
- definition
- occurs in what phase of cell cycle
- cause
- recognized/excised by
- repaired by
- = loss of an amino group from cytosine that converts a cytosine to a uracil.
- The uracil is recognized and removed (base excision) by a uracil glycosylase enzyme
- subsequently, this area is recognized by an AP endonuclease that removes the damaged sequence from the DNA
- occurs during the G1 phase of the cell cycle
- caused by: spontaneous/heat/nitrates (i.e. hotdogs)
- recognized/excised by: uracil glycosylase, AP endonuclease (removes deaminated cytosine)
- DNA polymerase (replaces missing pieces of sequence: fills in gap by synthesizing DNA in the 5’ to 3’ direction, using the undamaged strand as a template) and DNA ligase repair strand (DNA ligase makes phosphodiester bond = type of covalent bond)
excision endonuclease
- makes nicks in the phosphodiester backbone of the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide
WHERE do MSH1 and MSH2 function in the cell cycle and what do they do?
- mismatch repair genes
- active in G2
baby was being treated for a bacterial infection and now presents with blue lips, nail beds and skin (cyanosis) and low BP
- Gray baby syndrome. Can result in death
- Tx: Stop Chloramphenicol immediately. Exchange transfusion may be required to remove the drug. Sometimes, phenobarbital (UGT induction) is used.
What genes control entry into S phase?
Rb and p53
6yo child brought to clinic bc parents were concerned with excessive lesions and blistering in face/neck area. lesions don’t go away w. typical ointments and creams and often become worse when the child is exposed to sunlight.
- Diagnosis?
- what else can you expect to see on this child?
- xeroderma pigmentosum
- AR disorder
- excision endonuclease deficiency
- expect to see excessive freckling throughout the child’s body
- also expect to see slight stature and poor muscle tone
- can diagnose by measuring the relevant enzyme excision endonuclease in white cells of blood
microsatellite instability
- microsatellites = short tandem repeats = di-, tri-, and tetranucleotide repeats dispersed through the DNA, usually in non-coding regions
- if cells lack mismatch repair, the replicated DNA will vary in the number of repeats at a certain location
- this variation = microsatellite instability
When making RNA,
- DNA is read ? to ?
- RNA is MADE ? to ?
When making RNA,
- DNA is read 3’ to 5’
- RNA is MADE 5’ to 3’
RNA polymerase moves in the ? to ? direction along the template strand of DNA
RNA polymerase moves in the 3’ to 5’ direction along the template strand of DNA
heterogenous nuclear RNA
- aka hnRNA or pre-mRNA
- found only in the nucleus of eukaryotic cells
- represents precursors of mRNA, formed during its post transcriptional processing
small nuclear RNA
- snRNA
- only found in the nucleus of eukaryotes
- one of its major functions is to participate in splicing mRNA (removal of introns)
ribozymes
- RNA molecules with enzymatic activity
- found in both prokaryotes and eukaryotes
RNA polymerase I
- one of 3 eukaryotic RNA polymerases
- located in the nucleolus and synthesizes 28S, 18S, and 5.8S rRNAs
RNA polymerase II
- one of 3 eukaryotic RNA polymerases
- located in the nucleoplasm and synthesizes hnRNA/mRNA and some snRNA
- inhibited by alpha-amanitin (mushrooms)
RNA polymerase III
- one of 3 eukaryotic RNA polymerases
- located in the nucleoplasm and synthesizes tRNA, some snRNA, and 5S rRNA
All transcription can be inhibited by
All transcription can be inhibited by actinomycin D.
- it binds to the DNA, preventing transcription
RNA polymerase II is inhibited by
RNA polymerase II (eukaryotic) is inhibited by alpha-amanitin (a toxin from certain mushrooms)
Prokaryotic RNA polymerases
- there is a single RNA polymerase that synthesizes all types of RNA in the cell
- its subunit structure is alpha2, beta, beta’
- a protein factor called sigma is required for the initiation of transcription at a promoter
- termination of transcription sometimes requires a protein called rho factor
- prokaryotic RNA polymerase is inhibited by rifampin
- actinomycin D binds to the DNA, preventing transcription
The coding strand in transcription is NOT used during transcription.
- what is another name for the coding strand?
- It is identical in sequence to ??
- coding strand aka antitemplate strand
- it is identical to the RNA molecule, except the RNA contains U instead of T (only in DNA)
proteins are made from ___ end to ___ end
amino end to carboxy end
DNA template strand is _____ to mRNA
DNA template strand is complementary and antiparallel to mRNA
essential amino acids
- there are 10 essential AA in kids (or an adult with PKU)
- arginine (only essential in kids; kids have + nitrogen balance and need more Nitrogen)
- histidine
- isoleucine
- leucine
- lysine
- methionine
- phenylalanine
- threonine
- tryptophan
- valine
“PVT TIM HALL”
- P is NOT PROLINE
- T is NOT TYROSINE
- there are only 9 essential AA in a normal adult
Nitrogen balance
nitrogen balance is the normal condition in which the amount of nitrogen incorporated into the body each day exactly equals the amount excreted
negative nitrogen balance
- occurs when N loss exceeds incorporation and is associated with:
- protein malnutrition (kwashiorkor; these patients present with edema)
- a dietary deficiency of even one essential AA
- starvation
- uncontrolled diabetes
- infection
positive nitrogen balance
- when the amount of N incorporated exceeds the mat excreted. associated with:
- growth
- pregnancy
- recovery phase of injury/surgery
- recovery from condition associated with negative N balance
Protein breakdown occurs in 2 cellular locations
- lysosomal proteases digest endocytosed proteins
- Proteasomes (large cytoplasmic complexes) digest older or abnormal proteins that have been covalently tagged with ubiquitin for destruction
AA that have R groups with + charge @ physiologic pH
lysine and arginine
AA that have R groups with a negative charge @ physiologic pH
aspartic acid, glutamic acid
AA that is an excellent buffer @ physiologic pH
histidine
Enzymes ______ energy of reaction, delta G
- Enzymes DO NOT EFFECT energy of reaction, deltaG
- enzymes LOWER energy of ACTIVATION
Vmax
= maximum velocity with a specified amount of enzyme
Km
= [substrate] required to produce half of the Vmax
Competitive inhibitors _____ Km
competitive inhibitors increase Km
There are how many hydrogen bonds between C and G?
There are 3 hydrogen bonds connecting C and G
glycosylases
- digest carbohydrates
- found in lysosomes
alpha 1 antitrypsin
protein synthesized primarily by the liver and secreted in the bloodstream
- its function is to protect cells by serving as an inhibitor of proteases released during a normal inflammatory response
- among the >90 variants of the gene, the Z and S variants are most often encountered with this deficiency.
- both are the result of point mutations, which can be detected using PCR
- a ZZ mutation causes the alpha 1 antitrypsin protein to misfiled and aggregate in the ER where it damages cells, and eventually causes cirrhosis.
normal levels of alpha 1 antitrypsin
90-225 mg/dL
noncompetitive inhibitors ______ Vmax
noncompetitive inhibitors decrease Vmax
Na+ range
136-145 mEq/L (Normal = 140)
K+ range
3.5-5 mEq/L (Normal = 4)
Cl- range
100-106 mEq/L (Normal = 104)
HCO3- range
22-26 mEq/L (Normal = 24)
BUN range
8-25 mg/dl (Normal = 15)
Cr (creatinine) range
0.8-1.2 mg/dl (Normal = 1)
Glucose range
60-100 mg/dl (Normal = 80)
osmolar gap definition and normal value
- difference btw the measured osmolality and estimated osmolality.
- Normal = less than 10
Things that elevate osmolar gap
- ethanol, methanol, ethylene glycol, acetone, mannitol
- Thus, an inebriated patient has an elevated osmolar gap
equation to calculate osmolar gap
2(Na) + (Glucose/20) + (BUN/3)
technically:
2(Na) + (Glucose/18) + (BUN/2.8)
T differs structurally from U how?
T has a methyl group -CH3
The base on a nucleoside is added where on the sugar?
Nucleoside = base added to the 1’ C of a sugar
Nucleotide structure (where is what added?)
phosphate attached to the 5’ Carbon of a nucleoside
There are how many hydrogen bonds between A and T?
A = T; they’re joined by 2 hydrogen bonds
Z-DNA
- a rare left-handed double-helical form of DNA that occurs in G-C rich sequences
- the biological function of Z-DNA is unknown, but may be related to gene regulation
What bonds are broken during denaturation?
- H bonds and base-stacking are disrupted during denaturation
- denaturation does NOT break covalent bonds
semi-conservative replication definition
- during DNA replication, the 2 complementary strands of parental DNA are pulled apart and each of these parental strands is then used as a template for the synthesis of a new complementary strand.
- each daughter cell has one new strand and one original strand of DNA
order of enzymes involved in DNA replication
- Helicase breaks the H bonds holding the base pairs together (allows DNA to unwind)
- single-stranded DNA binding protein (SSB) stabilizes SSDNA to prevent it from returning to DSDNA or being degraded by nucleases
- Primase creates a primer ( approx 10 nucleotides) in 5’ to 3’ direction. (this is repeated for each okazaki fragment)
- DNA polymerase III synthesizes DNA in 5’ to 3’ direction
- steps 3 and 4 are repeated for each okazaki fragment
- in Eukaryotes: RNA primers are removed by RNAase H and a DNA polymerase fills in the gap that’s left with new DNA
- in Prokaryotes: DNA polymerase I both removes the primer (with its 5’ exonuclease activity) and fills in the gap with new DNA
- DNA ligase seals the nicks between okazaki fragments
- DNA gyrase (DNA topoisomerase II) proviees a swivel in front of each replication fork.
- As helicase unwinds the DNA the area in front of the replication fork becomes positively supercoiled (more tightly wound)
- DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands
DNA repair during G1 phase of EUKARYOTIC cell cycle
- UV radiation –> thymine (pyrimidine) dimers; fixed by excinuclease
- deaminations: C –> U; uracil glycosylase
- loss of purine or pyrimidine; AP endonuclease
DNA repair during G2 phase of EUKARYOTIC cell cycle
- mismatch repair: hMSH2, hMLH1
- both lead to HNPCC
4 bacterial causes of bloody diarrhea
SSEY
- Salmonella
- Shigella
- E.Coli
- Y. Pestis
Eukaryotic Ribosome
- total
- subunits and pieces in each
- 80S
- 60S subunit: 5S RNA and 5.8S, 28S RNA
- 40S Subunit: 18S RNA
Shiga toxin (shigella dysenteriae) and verotoxin (a shiga-like toxin in enterohemorrhagic E.Coli) inactivate the ______
- Shiga toxin (shigella dysenteriae) and verotoxin (a shiga-like toxin in enterohemorrhagic E.Coli) inactivate the 28S rRNA in the 60S subunit of the eukaryotic ribosome
- the A subunits of these toxins are RNA glycosylases that remove a single adenine residue from the 28S rRNA
- this prevents ammoniacal tRNA binding to the ribosome
- this halts protein synthesis
- this can result in bloody diarrhea
Name some diseases that result from errors in transcriptional processing:
- beta thalassemia
- SLE
- Tay- Sachs
- Gauchera
- Type IIA hyperlipidemia (bc a LDL receptor is mis-spliced)
- Marfans
how can 2 different proteins have the same mutation at the same site?
alternative splicing; these came from the same pre-mRNA strip
Prokaryotic Ribosome
- total
- subunits and pieces in each
- 70S
- 50S Subunit: 5S RNA and 23S RNA
- 30S Subunit: 16S RNA
newborns don’t have enough of what enzyme and as a result develop grey baby syndrome?
UDP-glucuronyl transferase activity
What is the start Codon?
AUG
what is the Eukaryotic equivalent of the shine-dalgarno sequence?
the 5’ cap; a methyl group is added to Carbon 7 of Guanine in mRNA at the 5’ end
activated amino acids are attached to the ____ on tRNA
activated amino acids are (covalently bonded) attached to the 3’ OH on tRNA
what is the smallest RNA?
tRNA
Chloramphenicol
drug given to fight bacterial infections, including meningitis
core enzyme of Prokaryotic RNA Polymerase consists of what subunits?
alpha 2, beta, beta’
Initiation of Prokaryotic Transcription
- Promoter consists of 2 parts: (-10) TATAAT and (-35 sequence)
- sigma initiation subunit required to recognize promoter
Termination of Prokaryotic Transcription
- stem and loop + UUUUUU (Rho independent)
- stem and loop + Rho factor
Initiation of Eukaryotic Transcription
- Promoter consists of 2 parts: (-25) TATA and (-70) CAAT
- Transcription factors (TFIID) bind promoter
- RNA polymerase binds to the promoter
- Where does polyadenylation of pre-mRNA occur?
- Why does it occur?
- the nucleoplasm
- generally associated with active gene expression in euchromatin
- the poly-A tail on eukaryotic mRNA protects the message against rapid degradation and aids in its transport to the cytoplasm.
The majority of cases of CF result from what genetic mutation? What does this cause?
- deletion of phenylalanine at position 508
- this results in a misfolded protein and the post-translational processing of oligosaccharide side chains
- result: abnormal CFTR (chloride channel protein) protein, that is then degraded by proteasomes
grey baby syndrome results from
babies do not have sufficient UDP-glucuronyl transferase activity needed to allow excretion of this chloramphenicol
- the drug builds up in the baby’s bloodstream and can lead to:
- blue lips, nail beds and skin (cyanosis)
- death
- low BP
cancers most often associated with Li-Fraumeni Syndrome
- cancers most often associated with Li-Fraumeni syndrome: breast, osteosarcoma, soft tissue sarcomas, brain tumors, leukemias, and adrenocortical carcinoma that affects the outer layer of the adrenal glands
- AD
- p53 gene inactivation or deletion
last 3 nucleotides in tRNA = ?
CCA
is Li-Fraumeni AD or AR?
AD
Rb gene binds to what?
- transcription factor E2F
- Rb gene acts as a tumor suppressor gene (negative regulator of the cell cycle) by binding to the transcription factor E2F and repressing transcription of genes required for S phase
- What has a 3’ to 5’ exonuclease?
- What has a 5’ to 3’ exonuclease?
- What has a 3’ to 5’ endonuclease?
- DNA polymerase has a 3’ to 5’ exonuclease activity for proofreading (The 3’ to 5’ can only remove one mononucleotide at a time)
- DNA polymerase 1 (removes prokaryotic RNA primers) has a 5’ to 3’ exonuclease (the 5’ to 3’ activity can remove mononucleotides or up to 10 nucleotides at a time.)
- the 3’ to 5’ endonuclease is used in processing of histone pre-mRNA
- What is deficient in xeroderma pigmentosum?
- AD or AR?
- excision endonuclease (which corrects thiamine dimers)
- AR
What does actinomycin D do and how?
- All transcription can be inhibited by actinomycin D.
- it binds to the DNA, preventing transcription
What is the function of the 5’ cap?
the 5’ cap is part of processing of eukaryotic pre-mRNA
the 5’ cap is part of processing of eukaryotic pre-mRNA
- the 7-methylguanosine cap is added to the 5’ end while the RNA molecule is still being synthesized. It serves 2 purposes:
1. serves as a ribosome-binding site
2. helps to protect the mRNA chain from degradation
- The poly-A tail attached where in processing of eukaryotic pre-mRNA?
- What is the poly-A signal?
- the poly-A tail is attached to the 3’ end of eukaryotic pre-mRNA
- AAUAAA
How is a poly-A tail attached?
- an endonuclease cuts the pre-mRNA on the 3’ side of the sequence AAUAAA (the poly-A signal)
- poly-A polymerase adds the poly-A tail (about 200As)
What is the purpose/function of the poly A tail?
the poly-A tail on eukaryotic mRNA protects the message against rapid degradation and aids in its transport to the cytoplasm.
*a few mRNAs (i.e. histone mRNAs) have no poly-A tails
- Where is the shine-dalgarno sequence?
- What does it do?
- only in bacterial cells
- at the 5’ untranslated region (UTR) of the message.
- helps ribosomes identify mRNA so they can interact
- protein synthesis begins at the AUG codon at the beginning of the coding region and continues until the ribosome reaches the stop codon at the end of the coding region
- Transcription and translation can occur simultaneously in bacteria, bc there is no processing of mRNA (no introns)
- Remember: RNA Pol READS 3’ to 5’ and transcription/translation occur 5’ to 3’
- The start codon is?
- what does the start code look like in DNA?
- AUG
- ATG (bc there is no U in DNA)
