Section III (Chapters 10-15) Flashcards
What the general sequence for signal transduction?
- Chemical messenger is secreted from specific cells in response to a stimulus
- Messenger diffuses/is transported to target cell
- Receptor in target cell specifically binds the messenger
- Binding of messenger to receptor elicits a response
- Signal ceases and is terminated
Endocrine signaling
Travel in blood, longer distancea
Paracrine signaling
Travel between nearby cells (think: synapse), specific to only nearby receptors (not every muscle cell with ACh receptors; just the neighbors)
What does paracrine signaling limit?
Immune response to specific locations within the body
Autocrine signaling
Act on same cell or sometimes same nearby cells
What type of chemical messenger does the nervous system have?
Neurotransmitters
Neurotransmitters
Contains nitrogen molecules (such as amino acids) or neuropeptides
Can be found at synaptic junctions or within the blood (neurohormones)
What type of chemical messengers does the endocrine system have?
Hormones
Hormones
Secreted from endocrine glands
Types: polypeptide hormones (insulin), catecholamines (epinephrine), steroid (cholesterol derived), thyroid (tyrosine derived)
What are specialized hormones that is not synthesized in endocrine cells?
Retinoids! -> derived from vitamin A & D
What chemical messengers are found in the immune system?
Cytokines
Cytokines
Small proteins that work to KILL invading microorganisms
Types: interleukins, tumor necrosis factors, interferons, colony-stimulating factors
Secreted via immune cells, and alter behavior of other cells by activating transcription of genes for proteins involved in immune response
Eicosanoids
Prostaglandins, thromboxane, and leukotrienes
Control cellular functions in response to injury
Derived from arachidonic acid (fatty acid), usually present as part of membrane lipid phosphatidylcholine
Growth factors
Polypeptides that function through stimulation of cellular proliferation or cell size
What type of receptor are nicotinic acetylcholine receptors?
Ligand-gated ion channel receptor
What occurs in the chemical synapse with nAChR?
Action potential causes changes in Na and K gradients across cell, resulting opening of Ca channel and influx of Ca which triggers fusion of vesicles within plasma membrane, releasing ACh into synaptic cleft, binds to nicotinic ACh receptor which contains a channel within the center of the receptors, causes conformational change opening the channel allowing Na to diffuse in and K to diffuse out which activates action potential down next nerve cell
(Sodium Potassium Pump!!!!)
What occurs in myasthenia gravis molecularly?
B & T lymphocytes develop pathogenic antibodies against nicotinic ACh receptors which bind to the receptors and cross-link them which is endocytosed and degraded
What does myasthenia gravis cause?
Inability of ACh to stimulate repeated muscle contraction (tirednesses utilizing muscle)
How do we test for myasthenia gravis?
Acetylcholinesterase -> edrophonium chloride
Should briefly improve the muscle weaknesses
Repetitive nerve stimulation and EMG can also show partial blockage of ion flux through muscular membranes
What are the treatments for myasthenia gravis?
Anticholinesterase agents
Immunosuppressive drugs
Thymectomy
Intravenous immunoglobulin
Plasmapheresis
Plasma membrane receptors
Spans plasma membrane containing extracellular binding domain
Types: Ion channel receptors, tyrosine kinase receptors, tyrosine kinase associated receptors, serine-threonine receptors, heptahelical receptors
What are the mechanisms for extracellular binding?
Phosphorylation of receptors, conformational changes in signal transducer proteins, or increases in levels of intracellular second messengers
What must the molecules not be able to do when using plasma membrane receptors?
Diffuse through cell -> polar molecules
What are intracellular receptors?
Utilized for messengers that diffuse into cell
Must be: hydrophobic
Mostly gene-specific transcription factors -> regulate transcription
Ion channel receptors -> nicotinic acetylcholine receptor
Similar to ACh receptors in neurotransmitters
Tyrosine kinase receptors -> insulin receptors
Present as single monomers that form dimers when messenger binds, Protein kinase transfers a phosphate group from ATP to hydroxyl group of amino acid (tyrosine), which is activated when the messenger binds to extracellular domain, causing phosphorylation of intracellular domain or associated protein, and the message is then propagated downstream through signal transducer proteins
G-protein coupled receptor -> glucagon receptor
Contain seven membrane spanning a-helices, when messenger binds to receptor, second messenger such as cAMP accumulate intracellularly, and transmit message
What must be looked over before the quiz?
ras-MAP kinase pathway & insulin receptor pathway
ras-MAP kinase pathway (in words)
One domain of receptor forms binding site for SH2 domain proteins (Grb2), which undergoes conformational change that activates another binding site called SH3 domain, which binds SOS protein , which catalyzes exchange of GTP for GDP on Ras, causing conformational change in Ras that promotes binding of Raf, triggering phosphorylation cascade, resulting in alteration of gene transcription factor activity
Insulin receptor pathway (in words)
Present in membrane as preformed dimer with a and B subunits, B subunits autophosphorylate when insulin binds activating the receptor, which then binds IRS, creating binding sites for SH2 domains, which binds Grb2, leading to activation of Ras and MAP kinase, and binds PI3 which phosphorylates PI4,5 bis to form PI345 trisP, PDK and PKB are recruited and phosphorylated
Tyrosine kinase receptors & serine/threonine kinase receptors similarity
Both messengers recruit two monomers to form dimer
Tyrosine kinase receptors & serine-threonine kinase receptors difference
Tyrosine kinase receptors binds type II receptor first which recruits type I receptor which is then phosphorylated by the type II receptor; then binds Smad protein which is phosphortylated at serine residue, undergoes conformational change and dissocates, then forms complex with Smad 4 or CoSmad forming Smad Complex which travels to nucleus to alter gene transcription
Where is cAMP synthesized from?
ATP
What pathway is this?
G-protein coupled signal transduction pathway
Steps for G-protein coupled signal transduction pathway
If target protein is adenylyl cyclase;
1. cAMP is synthesized from ATP
allosteric activator of PKA, which phosphorylates CFTR, phosphorylase kinase, and phospholmaban, and transcription factor CREB
2. cAMP is hydrolyzed to AMP by cAMP phosphodiesterase
3. Some heptahelical receptors bind q isoform of Ga subunit, activating phospholipase CB, which hydrolyzes membrane lipid PI 45 Bis into DAG and IP3
4. IP3 has binding site in SR and ER stimulating Ca release and activation of calcium-calmodulin subunits
5. DAG remains in membrane and activates protein kinase C, which propagates response by phosphorylating target proteins
What secondary messengers are produced by adenylate cyclase?
cAMP -> activates PKA
What are the two heterotrimeric G protein systems?
Adenylate cyclase & phospholipase C
What secondary messengers are produced by phospholipase C?
IP3 -> stimulates Ca release and activates Ca-calmodulin subunits
DAG -> activates PKC
What are the different ways to terminate signals?
Phosphorylation
Internalization
Degradation
Automatic termination of messages due to hydrolysis of GTP
Degradation of secondary messengers
How does insulin work?
Via tyrosine kinase receptors to promote fuel storage
What does epinephrine promote?
Fuel mobilization
What is the action of guanylyl cyclase receptors?
Convert GTP to second messenger cGMP (analogous to cAMP) directly, unlike heptahelical receptors which use G protein to signal adenylyl cyclase to produce cAMP; activates protein kinase G, phosphorylates targets
What do we use guanylyl cyclase receptors for?
Angina pectoris, heart failure, and erectile dysfunction
What occurs in cholera?
Watery diarrhea leading to dehydration and hypovolemic shock caused by cholera toxin ADP-ribosylating a class of G-proteins, altering their function and affecting water and salt transport across the intestinal mucosa.
Treatment: glucose electrolyte solution to increase coupled glucose sodium uptake into intestinal epithelial cells, reversing the loss of water from these cells
What occurs in anorexia nervosa?
Effects of inadequate nutrition on hormone release and response. Cortisol, glucagon, and epinephrine levels are all increased under these conditions
What are the basic principles of signal transduction?
Cells receive and respond to a number of different signals at any given time
Only cells with receptors to a specific signal (chemical messenger) will respond to that signal
One signal can elicit different effects in different cell types
When is the signal transduced?
Binding of a chemical messenger to its receptor, amplified in the process
What occurs when insulin binds to receptors on hepatocytes?
Glycogen synthesis, fatty acid synthesis, and cholesterol synthesis
What occurs when insulin binds to receptors on skeletal muscle fibers?
Stimulate glucose uptake, glycogen synthesis, and other anabolic processes
What is within microchondria?
less than .1% of genome DNA, similar to bacteria/prokaryotes, not enclosed within membranes
What is within viruses?
contain either DNA or RNA genome, proteins required for pathogenesis or replication, and protein coat, but lack complete DNA systems of replication (must invade and commandeer host DNA)
What is ribose?
RNA sugar
What is deoxyribose?
DNA sugar
Where is the poly A tail?
At the 3’ end of DNA
What are the major & minor grooves?
Alternating size around outside in DNA
What is chromatin?
DNA complex bound to proteins (nucleosomes)
What does complementary mean?
Opposite matching strand sequences
What does anticodon mean?
Trinucleotide sequence on tRNA
What does gene mean?
DNA sequence that encodes components of gene product with regulator
What does hybridization mean?
Reannealing/renaturation of DNA strand with self or with RNA
What does ribosome mean?
Site of translation, contains rRNA & tRNA
What does histone mean?
Proteins that DNA winds around to condense in nucleus
What does double helix mean?
General shape of DNA
What does codon mean?
Binding spot on mRNA, where tRNA binds to direct translation
What does nucleosome mean?
Clusters of histones forming beads on string formation
What does antiparallel mean?
Two strands running opposite direction 5 to 3 and 3 to 5
What does nucleotide mean?
Sugar, base, and phosphate
What does nucleoside mean?
Sugar & base
What are the base pairings
A - T
G - C
What is the basic structure of a nucleotide?
Heterocyclic nitrogenous base, sugar, and phosphate, linked via 3’ to 5’ phosphodiester bonds
What is the general structure of DNA?
Double-stranded with two antiparallel polynucleotide strands, joined via hydrogen bondings within base pairs
Strands run in opposite directions of one another (one 3’ to 5’ and one 5’ to 3’)
What is the importance of base pairing, hydrogen bonds, van der Waals interactions, and the hydrophobic effect in DNA?
Base pairing is specific and occurs by hydrogen bonds between two strands
Van der waals and hydrophobic effects stabilize the molecule in stacking of the base pairs
What can cause the denaturation of DNA?
Alkali solutions and heat
What do alkali solutions do to DNA?
They do not break phosphodiester bonds of DNA, but rather cleave RNA due to hydroxyl group on 2’ carbon losing proton
What does heat do to DNA?
Tm represents the temperature in which DNA is 50% seperated
Can be realigned in temperature is lowered!
Prokaryotic DNA
Packaged in doubled stranded circle with about 4x10^6 bp, fairly large, 3000 genes
Eukaryotic DNA
Packaged in nucleosomes and wound into chromatin, approximately 6 x 10^9 up to 25,000 genes (much bigger!)
What is within a histone?
Large amounts of arginine and lysine, binds to DNA to form chromatin
Histone clusters form…
Beads on a string formation known as nucleosomes, with 4 x2 histones forming core and a fifth type of histone H1 complexing with the DNA joining the cores
Where are DNA found?
Within nucleus in nuclear envelope and in mitochondria via primordial eukaryotic cells
What are plasmids?
Small circular DNA molecules that can enter bacteria and replicate autonomously; can be used as tools for microbiological transfer of genes
What is the structure of DNA?
Bound to proteins forming chromatin, euchromatin, or heterochromatin
Contains double ring purines AG, single ring pyrimidines CT, and a deoxyribose sugar
Negatively charged due to free OH on phosphate dissociating a H ion
Nucleosides are linked via…
N-glycosidic bond to anomeric carbon of sugar
Nucleotides are linked via…
The phosphate attached to 5’ hydroxyl group of sugar in ester linkage
What is the function of DNA?
Purine on one strand forms hydrogen bond with pyrimidine on other
One strand of DNA can serve as template for synthesis of other strand
Also allows for DNA to serve as template for synthesis of complementary strand of RNA
Where are RNA located?
Formed in nucleolus, moving to specialized regions of cytoplasm depending on the type of RNA formed
What is the structure of RNA?
Similar 3’ to 5’ phosphodiester bonds forming strands, however uracil replaces pyrimidine thymine
Sugar is ribose instead of deoxyribose, ribose contains hydroxyl group on 2’ carbon , which makes it susceptible to alkaline hydrolysis
Single stranded and lack helical structure, though secondary and tertiary structure can occur due to strand looping back on self
What is thymine replaced with in RNA?
Uracil
What are the functions of RNA?
mRNA, rRNA, tRNA participate in process of protein synthesis
Define mRNA
Contain nucleotide sequence converted to amino acid sequence of protein in process of translation
What is contained with mRNA?
contains 5’ cap at end, poly A tail at 3’ end, and coding region between
5’ end has leader sequence with guanosine cap and then later start codon
3’ end contains poly A tail that may be up to 200 nucleotides long
Guanosine cap and poly a tail are posttranscriptionally added
Define rRNA
Has extensive internal base pairing and complexes with proteins to form ribosomes, which bind mRNA and tRNA during translation
Define tRNA
Activates specific amino acid for insertion into growing polypeptide during translation
What is unique about tRNA?
unique trinucleotide sequence on each tRNA is anticodon which binds to complementary codon on mRNA
cloverleaf structure
What is the central dogma?
Information flows from DNA to RNA to proteins
What is the exception of the central dogma?
Retroviruses!
Invade cells, RNA genome is transcribed to produce DNA copy, using reverse transcriptase
What do we use 5-FU in?
Cancer treatment
- a pyrimidine base similar to uracil and thymine, which inhibits the synthesis of thymine nucleotides by inhibiting thymidylate synthase as a transition state analog, which normally converts deoxyuridine monophosphate to deoxythymidine monophosphate, which ceases DNA synthesis and therefore cell proliferation in cancer
What do we use ZDV in?
Viral replication
- nucleoside analog reverse transcriptase inhibitor, acts as a thymine nucleotide that when attached to growing 3’ end of DNA, due to lacking 3’ OH group, DNA synthesis is terminated, terminating viral replication
Why does azithromycin work for bacterial infections without harming the host?
Azithromycin is a macrolide antibiotic
Binds to bacterial rRNA stopping protein synthesis by inhibiting transpeptidation/translocation of protein
What are the side effects of azithromycin?
Because it can inhibit mitochondrial protein synthesis, can lead to epigastric distress, diarrhea, or cholestatic jaundice
If there is metastasis from colon to liver, what is palliative?
5-FU therapy
What must retroviruses do?
Invade host cells
What occurs in AIDS?
Infection by the HIV, a retrovirus containing an RNA genome. Through its growth in immune cells, active infection by the virus leads to an immunocompromised state.
Nucleoside analogs are one class of drugs used to treat people with HIV infections
What occurs in adenocarcinoma?
Use of nucleotide analogs as chemotherapeutic agents. Specifically, 5-fluorouracil is used to inhibit deoxythymidine monophosphate (dTMP) synthesis by the enzyme thymidylate synthase, which leads to the death of rapidly proliferating cells
What occurs in pneumonia?
A bacteria-induced illness in the lungs, leads to fever and cough. Treated with anitbiotics. Certain antibiotics target bacterial protein synthesis but may also inhibit mitochondrial protein synthesis
DNA synthesis in prokaryotes
DnaA protein binds at point of origin (oriC) where DNA coils around DnaA core
With assistance of helicase, gyrase, single stranded binding protein, two parental strand separate
Synthesis occurs at two replication forks that move away from each other
Second round of synthesis can start before other is finished
DNA synthesis in eukaryotes
Replicates histones with DNA
Multiple points of origin, bubbles appear where fork can proceed in each direction
Many more polymerases and complex proteins
What does bidirectional mean in context of DNA?
Fork travels in both directions from origin
What does semiconservative mean in context of DNA?
Each daughter chromosome contains one parental strand and one newly synthesized
Origin of replication
Start point for prokaryote DNA synthesis where DnaA binds
Leading strand
Continuous 5’ to 3’ synthesis toward the fork
Lagging strand
Discontinuous 5’ to 3’ synthesis in short fragments called Okazaki, away from the fork, jointed together by ligase
Okazaki fragments
Short fragments of discontinuous synthesized DNA
Helicase
Unwind two parental strands
Topoisomerase
Unwind two parental strands by breaking phosphodiester bonds and rejoining them to relieve supercoiling (DNA gyrase in prokaryotes)
Single-strand binding protein
Prevents DNA from “reannealing”, or folding over onto itself, to protect from enzymes that cleave single stranded DNA
Primase
Synthesizes RNA primer molecule in a 5’ to 3’ direction that copies DNA template strand
RNA primer
Ribonucleotide sequence added for site where DNA polymerase can add to 3’ end, removed and replaced
PCNA
Proliferating cell nuclear antigen, organizes and orchestrates replication process on both leading and lagging
DNA polymerase
Copies each parental template 3’ to 5’ producing new strand 5’ to 3’
RNase H
Ribonuclease that specifically degrades RNA from RNA-DNA hybrid, can also assist with removal of primers
Ligase
Joins Okazaki fragments, free 3’ OH to phosphate at 5’ end
What phase of the cell cycle does DNA replication occur?
S-phase of cell cycle, followed by G2 phase then M, where cell divides creating daughter cell with exact copy of DNA
Why are the ends of linear chromosomes lost during replication?
Primase cannot lay down a primer at the very end of a chromosome
Results in 3’ overhang regions of template on new strand
Telomeres
Repeating sequences of bases TTAGGG
What is the purpose of telomerase in DNA?
An RNA dependent DNA polymerase (like reverse transcriptase), uses own RNA template to synthesis new repeating DNA to lengthen DNA overhang, primase can then bind at long end and synthesize back to end of coding regions, telomere binding proteins protect ends of chromosomes from damage and nuclease attack
What is the base-pairing correction in prokaryotes?
Pol III (a replication enzyme) performs proofreading, 3’ to 5’ exonuclease activity in addition to polymerase activity
Exonuclease activity: incorrectly base-paired nucleotides are removed before the growing chain is lengthened
Only 1 in a million error
What are the mechanisms of eukaryotic repair?
Nucleotide excision repair, base excision repair, mismatch repair, transcription-coupled repair
Nucleotide excision repair
Local distortions such as a mismatch in base pairs or damage to bases, specific endonucleases cleave abnormal chain removing distorted region, gap filled by DNA polymerase using intact strand as template, joined by ligase
Base excision repair
DNA glycosylases recognize a small distortion involving lesions caused by damage to single base, glycosylase cleaves N-glycosidic bond joining damaged base to deoxyribose, then endonuclease cleaves the sugar-phosphate strand at the site, then polymerase fills the gap, ligase joins
Mismatch repair
Mismatched bases recognized by enzyme complex during replication, removed and replaced
Transcription coupled repair
RNA polymerase that is transcribing a gene stalls when it encounters a damaged portion, excision repair proteins repair damaged region similar to NER
How do bacteria distinguish between new and parental strands?
Bacteria parental strands contain methyl groups on adenine bases, new strands are not immediately methylated
What damage occurs to DNA via UV light?
UV alter base pairing by exciting adjacent pyrimidine bases on DNA strands, causing them to form covalent dimers, usually thymine dimers
What DNA damage occurs via cigarette smoking (benzo[α]pyrene)
Benzo a pyrene coats airways and lungs, can cross cell membranes, and interact with DNA, causing damage to bases interfering with base pairing, specifically makes bulky adducts with guanine residues in DNA
What repair is done to UV light & cigarette smoking damaged DNA?
NER repair
What are genetic rearrangements?
Exchange of segments between DNA molecules
What are the 3 major mechanisms of genetic rearrangement?
Homologous recombination, translocations, transposons
Translocation
Chromosomes break and portions randomly become joined to others
Breakage by x-ray or carcinogen, and resealing of parts of different chromosomes
Transposon
Can move from one site in genome to another or produce copies that can be sent to new site
What is contained within transposons?
Contain gene for enzyme transposase, cleaves transposon from genome and moves to another location
What is occurring here?
Homologous recombination
Reverse transcriptase mechanism
RNA template is transcribed from DNA by RNA polymerase or obtained from RNA virus
RNA template forms cDNA strand, is degraded, and second cDNA strand formed
What is the mechanism of dideoxynucleoside NRTIs?
No hydroxyl group on either 2’ or 3’ carbon, can be converted to dideoxynucleoside triphosphates in cells, and then when incorporated into growing DNA, termination results due to lack of 3’ hydroxyl group
Why can HIV genome rapidly mutate?
Reverse transcriptase (the enzyme that converts single stranded RNA to DNA) lacks proofreading ability, so it can mutate at a rapid rate
What is faulty in xeroderma pigmentosum?
Defect in NER or transcription coupled repair, sun hypersensitivity with high risk of skin cancer, inability to remove thymine dimers
What is faulty in cockayne?
Transcription coupled repair defect, premature aging, premature cell death due to reduction of gene expression, because damaged genes cannot be translated
What is faulty in HNPCC?
Mutation in enzyme present in intestinal epithelial cells responsible for mismatch repair, leads to colorectal cancers
What is faulty is hereditary breast cancer?
Inheritance of mutations in proteins responsible for DNA repair of single strand and double strand breaks
What does diabetes put patients at risk for?
Infections due to high glucose levels -> perfect culture medium for bacteria
Define transcription
Synthesis of RNA from DNA template
Define template strand
DNA strand that directs sequence of nucleotides
Define coding strand
Nontemplate strand, will be identical to RNA transcript except T changed to U, determines protein sequence
Define promoter sequence
DNA sequence that defines where gene transcription begins
Define primary transcript (hnRNA)
Initially generated RNA strand from transcription, receives guanosine cap at 5’ end and poly A tail at 3’ end
Define upstream in RNA synthesis
To left of start point, or towards 5’ end
Define downstream in RNA synthesis
To right of start point, or towards 3’ end
Introns
No coding functions, removed during splicing
Exons
Expressed part, contain coding sequences for proteins
What is the fun thing Aloki likes to say for introns and exons
Exons we keep, introns we yeet
Coding strand is … to RNA transcription produced, RNA strands dictate… chain
Identical, protein
Template strand is … strand that has base pairs bond in matching order to … strand
Actual, coding
How is RNA polymerase different from DNA polymerase?
Initiate new synthesis without primer
RNA polymerase … polymerization of ribonucleotides
Catalyzes
What must RNA polymerase be able to do?
Recognize promoters for transcription starting site
RNA polymerase lacks…
3’ to 5’ endonuclease activity (but has some rudimentary error checking)
Prokaryotic RNA polymerase
Single RNA polymerase that transcribes DNA to generate all different types of RNA
Five subunits with sigma factor that binds and directs promoter binding
Eukaryotic RNA polymerase
Three different RNA polymerases, I = rRNA, II = mRNA and microRNA, III = tRNA and 5S rRNA
Different promoters
What is a promoter?
Composed of boxes or elements that are contiguous with transcribed region, control binding of RNA polymerase and to DNA, identifying start point
-10 region in prokaryotes is the
TATA box
-25 region in eukaryotes is the
TATA box
-35 region in prokaryotes is the
TTGACA sequence
What do eukaryotes frequently have?
GC rick sequence/BRE, initiator element, downstream promotor, motif ten element
What are the proximal promoters regions in eukaryotes?
-100 to -200
First base of coding strand is …, untranscribed sequence in 5’ flanking region are numbered … and so on
+1, -1
What is the role of sigma factor in prokaryotic transcription?
Binds to RNA polymerase enzyme to direct promoter binding
Binds to TATA box region of promoter, sigma 70
What are the 2 mechanisms of termination for sigma factor?
Transcription termination signal of either hairpin loop preceding several U residues, or binding of a rho factor protein which causes release of RNA transcript from template
Polycistronic transcripts
Contains information to produce several different proteins
Usually common in bacteria, several different proteins formed at once (operon)
Monocinstronic transcripts
Contains information for one protein
Usually common in eukaryotes, requires introns removed and other posttranscriptional modifications
What are the posttranscriptional processing events?
5’ end capped as it is transcribed: first loses a phosphate becoming a diphosphate which attacks GTP
Methylated guanosine = CAP 0
Addition of methyl group to 2’ carbon = CAP1
Addition of another methyl group to next nucleotide 2’ carbon = CAP2
Methyl groups donated by SAM
3’ end acquires poly A tail
Removal of introns: introns = “nonsense information”: intron barriers, spliceosome
What is the importance of splicing eukaryotic transcripts?
Consensus sequences at intron/exon boundaries are AGGU which vary to some extent on exon side of boundary, but intron side are fairly invariant
Spliceosome complex forms loop formation cutting out intron, and linking exons together
Posttranscriptional processing for tRNA
cloverleaf shape that folds into 3D L shape
start with 100 nucleotide precursors off of RNA strand, 5’ and 3’ ends are cleaved
introns removed by endonucleases
uracil methylated by SAM to form thymine
one of double bonds of uracil is reduced to form dihydrouracil
uracil residue is rotated to form pseudouridine
Posttranscriptional processing for rRNA
large gene exists in many copies in nucleolar organizer region of nucleus
produces large 45S transcript which can be cleaved to form 18S, 28S, and 5.8S
can become methylated which may signal for cleavage
What is the difference between eukaryotic and prokaryotic DNA & RNA
Eukaryotes have introns, repetitive sequences, histones, and errors occur in nucleus
Prokaryotes are open ring, and errors occur in cytosol
What occurs in beta-thalessemia?
Decrease or abolish the synthesis of a or B chains in adult hemoglobin A tetramer, which interfere with transcription of B globin transcription, processing, or translation
Mutation within TATA box for B globin gene, reducing accuracy of start point, so only portion of normal protein synthesized, other mutations further upstream in promoter region
Or
Mutation in promoter region of B globin gene, results in significantly decreased rate of synthesis in homozygous
Minor, intermediate, or major: amount of B globin gene affected, which then affects total hemoglobin level = anemia
What is the difference between beta-thalassemia and sickle cell anemia?
Beta-thalassemia does not cause muscle pain with exacerbation during exertion
Mechanism of action of transcription inhibitors rifampicin and alpha-amanitin
Inhibits eukaryotic RNA polymerases, especially polymerase II
Lack crucial OH group, so chain cannot be continued after addition to chain
What is the genetic code?
Groups of 3 nucleotides that code for an amino acid = a codon. There are 64 of these.
What are anticodons?
Adapters on tRNA that bind directly with an mRNA codon, transferring an amino acid to the growing polypeptide chain
What occurs in transcription?
The transfer of genetic message from DNA to RNA
What occurs in translation?
The transfer of genetic message from nucleic acids to amino acids
What occurs in replication?
The replication of genetic material in DNA
Why is the genetic code degenerate, unambiguous, nonoverlapping, and almost always universal?
Many amino acids are specified by more than one codon = degenerate
Each codon only specifies one amino acid = unambiguous
No extra nucleotides or punctuation to separate codons- each nucleotide is only read once = nonoverlapping
What is always the coding strand unless stated?
DNA
Point mutation
One base in DNA altered producing change in signle base of mRNA codon
What are the types of point mutation?
Silent, missense, nonsense
Missense mutation
Amino acid is replaced by other
Nonsense mutation
Change to stop codon causing premature termination
Silent mutation
Does not impact amino acid
Insertion
One or more nucleotides are added to DNA, if stop codon not generated, a protein with more amino acids could be produced
Deletion
One or more nucleotides added to DNA, if deletion does not affect normal start and stop, shorter protein can be produced
Frameshift
Number of inserted or deleted is not multiple of three, causes reading frame to shift, beyond point of mutation, amino acids will differ from normal protein
What do anticodons on tRNA match with?
Three base sequence of codons on mRNA strand
What does each tRNA carry?
Specific amino acid, thus amino acids are linked in sequence according to mRNA codons
Three phases of translation
Initiation, elongation, and termination
Initiation
Formation of a complex containing methionyl tRNA bound to AUG start codon of mRNA and to P site of ribosome, requires GTP and eIFs
Methionine tRNA initially forms complex with IF2 which binds GTP which then binds to ribosome with participation of IF3
IF4F binds 5’ cap
Elongation
Binding of aminoacyl tRNA to A site on ribosome base pairing with second codon on mRNA, formation of peptide bond between first and second amino acid, translocation of mRNA relative to ribosome so third mRNA codon moves into A site
GTP must be hydrolyzed for tRNA to stay in A site
Termination
Termination codon aligns with site on ribosome where next aminoacyl tRNA would normally bind, release factors bind instead causing completed protein to be released from ribosome
How is the correct 3D conformation of polypeptide achieved?
Proteins begin folding as they exit the ribosome
Chaperone proteins help with folding
Disulfide isomerases help disulfide bond formation between cysteines
Posttranslational modifications on amino acids
Cleavage of methionine or other to form new N terminal amino acids
Acylation of N terminal amino acid
Methylation of lysine
Hydroxylation of proline and lysine
Carboxylation of glutamate
Fatty acylation and prenylation
Transfer of ADP ribose group from NAD+
Phosphate addition or removal
Glycosylation
What is the mechanism for protein targeting?
Targeting sequences or signal sequences: amino acid sequences that facilitate transport to certain organelles if synthesized in cytosol
Proteins synthesized bound to RER are destined for secretion to organelles or cellular membranes
What do the proteins synthesized bound to RER contain?
Contain signal peptides near N terminus that do not have common amino acid sequence, with several hydrophobic residues , SRP binds and attaches to SRP receptor in rough ER, signal peptide removed by signal peptidase, then transferred in small vesicles to golgi, golgi sorts and processes proteins such as glycosylation and modification
What do the mutations cause the beta-globin transcript to do?
Function, shape, and size of beta globin protein is impacted
What leads to Tay-Sachs disease?
Insertion within the alpha-chain of hexosaminidase A gene
Causes dysfunction of hexosaminidase enzyme which normally degrades glycosphyngolipids like gangliosides
Dysfunction leads to build up of gangliosides and neurological symptoms, vision difficulty
What is an E6V mutation?
Missense mutation that impacts hemoglobin -> sickle cell
Valine residue replaces glutamic acid at position six of the beta-chain
What occurs in I-cell disease?
Lysosomal proteins are not sorted properly in golgi and up being secreted from cell, due to mutation of phosphotransferase enzyme which is a required first step to attach lysosomal targeting signal m6p, organelles become clogged with materials that cant be digested, lysosomes swell because enzymes not present to degrade
What does streptomycin inhibit?
Inhibits translation initiation by binding to three proteins and ribosomal subunit 16S of bacteria
What does tetracycline inhibit?
Binds to 30S ribosomal subunit of bacteria and prevents aminoactyl tRNA from binding to A site on ribosome
What does chloramphenicol inhibit?
Binds to ribosomal subunit 50S and prevents binding of amino acid portion, inhibiting peptidyltransferase action
What does erythromycin inhibit?
Bind to 50S ribosomal subunit near binding site for chloramphenicol, however prevents translocation step so tRNA cannot move from A to P
What is the need for cells to regulate expression of its genes?
Efficiency and ability to express specific proteins, and ability to adapt to changes in environment
What is an operon?
Genes of a related function are grouped sequentially and coordinately expressed (all on, or all off)
Why is an operon of genes well-suited for regulation of gene expression for prokaryotes?
Single polycistronic mRNA produced coding for all proteins of the operon
Allows more adaptability
Where does a repressor protein bind?
Operator blocking transcription
What is an inducer?
Stimulates expression of operon by binding to repressor and changing conformation causing release from operator (nutrient or metabolite of nutrient- lac operon)
What is a corepressor?
Repressor is inactive until corepressor binds which activates it allowing it to bind to the operator preventing RNA polymerase (if product is available, save energy by not transcribing more – trp operon)
What is the negative control within the lac operon?
Repressor binding to operator
What is the positive control within the lac operon?
Allolactose binding to repressor
How does the existence of different sigma factors that bind RNA polymerase result in prokaryotic gene regulation?
Stimulates binding to certain promoters, activating transcription of specific operons, thus specific gene expression
Transcriptional attenuation is
Interrupting transcription after it has been initiated
As mRNA is being transcribed, hairpin loop forms in mRNA due to speed of translation too high due to high availability of trp
Requires coupled transcription and translation so cannot occur in eukaryotes
Most regulations in eukaryotes is…
Positive
Very complex with complex functions within each cells that change as development continues, environments change, etc.
Genes must be active to be expressed, no operons/often located on different chromosomes, transcription and translation are separated
What is the role of chromatin remodeling?
Chromatin must remodel so that not part of a nucleosome, and chromatin is in euchromatin/loose state, not heterochromatin/dense
ATP driven chromatin remodeling complex which uses energy from ATP hydrolysis to unwind sections of DNA from nucleosome core
Covalent modification of histone tails through acylation by histone acetyltransferases, making it easier for DNA to unwind from histones
What is the role of DNA methylation?
Methylation of cytosine residues located in CG rich sequences, often near promoter regions
Usually less readily transcribed than those that are not methylated
What are gene-specific transcription factors?
Regulatory proteins that bind directly to DNA sequences
- Activators, inducers, repressors, nuclear receptors
What are hormone-binding transcription factors?
Steroid hormones that activate or inhibit the transcription of specific genes through binding to nuclear receptors
Nuclear receptors bind to DNA regulatory sequences called hormone response elements and induce or repress transcription
Basal transcription factors
Basal are bare minimum for transcription
Gene specific enhance basal model for faster expression of specific gene
Alternative splicing
splicing out of introns to form exon coding regions
Polyadenylation
poly A tail to 3’ end
mRNA editing
editing bases or addition or deletion of nucleotides after synthesis
What are the mechanisms of regulation at the level of translation and mRNA stability?
Initiation of protein synthesis by initiation factors Ifs, which are regulated through mechanisms involving phosphorylation
Transferring moves iron into cells (higher if levels low in blood)
Ferritin stores iron (less if iron levels low in blood)
How does a single RNA binding protein, IRE-BP, coordinates the levels of ferritin and the transferrin receptor to ensure that cellular and bodily iron levels are tightly regulated?
IRE-BP bind to transcript when low iron, high iron binds to IRE-BP to remove
Ferritin: IRE binds at 5’ end, blocks ribosome
Transferrin: binds to 3’ end to stabilize and prevent degredation
How can microRNAs (miRNAs) can posttranscriptionally regulate gene expression?
microRNAs can induce degradation of target mRNA or block translation of mRNA
miRNA -> pri-miRNA -> pre-miRNA -> mature miRNA -> incorporation into RISC complex -> RISC blocks translation of target mRNA
Non-Hodgkin lymphoma
Treatment with multiple drugs, all targeted to inhibiting cell proliferation, but through different mechanisms. DNA synthesis is targeted, as is tubulin action (to block cell division). DNA damage is induced, and thymidine synthesis is also blocked to inhibit further DNA replication
CML (chronic myelogenous leukemia)
More than 90% of CML arises owing to the generation of the Philadelphia chromosome, which is created by an exchange of genetic material between chromosomes 9 and 22. This translocation creates a unique fusion protein (BCR-abl), which facilitates uncontrolled proliferation of cells which express this fusion protein
Anorexia nervosa
The patient’s poor diet has led to a hypochromic anemia owing to low iron levels. This leads to a reduction of expression of serum and tissue ferritin, but an increase of expression of the transferrin protein and the transferrin receptor
Angelman and Prader–Willi syndrome
The use of base methylation, within promoter regions, to regulate gene expression. The methylation of key bases within the promoter leads to nonexpression of the gene and forms the basis for imprinting. This is an example of epigenetic modification of gene expression
Fragile X disease
A significant number of triplet repeat expansions within a gene may lead to dysfunction of the protein product, leading to disease. In fragile X, impairment of cognitive function is the primary symptom, caused by expansions in the FMR-1 gene on the X chromosome
Androgen insensitivity
Lack of androgen receptors, leading to default female sexual characteristics. The patient produces androgens but cannot respond to them. These patients have an XY genotype but female sexual characteristics
Diphtheria, pertussis
Diphtheria toxin catalyzes the ADP-ribosylation of eEF2, a necessary factor for eukaryotic protein synthesis. This results in cell death. Vaccination against pertussis antigens will prevent infection
Hutchinson–Gilford Progeria syndrome
An example of a silent mutation in terms of amino acid substitution, but the single nucleotide change creates an alternative splice site that leads to a loss of 50 amino acids from the precursor lamin A protein. This leads to altered posttranslational processing and the symptoms of a premature aging disease
