Midterm 2 Flashcards
Normal vs Poor Control of Blood Glucose
normal control has fasting levels around 5mM and small rapid spikes of blood glucose after meals.
poor control has fasting levels around 12mM and exaggerated spikes of blood glucose after a meal.
Burning Glucose
directly after a meal we burn glucose (glycolysis plus the TCA cycle at incredibly high rates to generate ATP and remove glucose from the blood). Requires oxygen.
Lowers the level of glucose in the blood.
Acetyl CoA - function and sources
substrate used in the TCA cycle to form ATP. Three ways to generate:
- glycogen to glucose through gluconeogenesis. then glycolysis to form pyruvate then oxidation to form acetyl CoA.
- triglyceride to free fatty acid through lipolysis. Beta-oxidation forms acetyl CoA. Can use free fatty acids anywhere but the brain. Can go in the reverse and synthesize free fatty acids from acetyl CoA.
- protein to amino acids by proteolysis then deamination and oxidation to form acetyl CoA.
Storage of Glucose
stored in the form of glycogen in the liver but can be rapidly converted by the liver into glucose which is then exported to the blood to maintain glucose levels in the fasted state.
liver can hold approximately 120 grams of glycogen
basal metabolic rate of glucose to CO2 is 10 grams per hour
have enough glycogen in liver for 12 hours.
Storage of Fatty Acids
stored as triglycerides (TAG or triacylglycerides) mainly in adipose cells. Fatty acid travels in the blood as two forms:
- free fatty acid which are available for uptake and metabolism
- TAG held in blood lipoproteins VLDL which is not readily available for uptake and metabolism.
lipolysis releases TAG to free fatty acids (remember 1 TAG contains 3 fatty acids)
everywhere but the brain can use free fatty acid to make acetyl CoA.
Storage of Amino Acids
stored mostly as protein in muscle. Average muscle protein has a half-life of less than a week so process of protein to amino acid and reverse is natural.
insulin stimulates growth and protein synthesis which aids in the recovery of muscle mass after fasting. Or if insulin is low (starved state) you accelerate breakdown of body protein to amino acids to acetyl CoA.
muscle protein is the most readily available for proteolysis to produce glucose in the starved state.
Glucose –> Fatty Acid
In the well fed state, insulin stimulates the liver to make fatty acids from glucose obtained from the diet by binding to receptors on the cell surfaces of the liver and adipose which causes activation of key metabolic enzymes that catalyze rate determining steps.
insulin also promotes localized release and uptake of fatty acid by adipose
Fatty Acids –> Ketone Bodies
Starved state. rising levels of catabolic hormones: epinephrine and glucagon. And falling levels of insulin.
hydrolization of TAG to release free fatty acids.
Brain cannot metabolize fatty acids for energy.
In the liver, ketone bodies can be formed. Cannot make glucose from fatty acids!!
Ketone Bodies
In the starved state, production of fatty acids from TAG are sent into the blood stream. In the liver, ketone bodies are formed which are released into the blood. Most cells can convert ketone bodies to acetyl CoA, and it is faster than using fatty acids to form acetyl CoA. Ketone bodies acidify the blood and give the person “ketone breath” which smells like acetone.
Well-Fed Phase: Hormones, fuels and results
Hormone Levels (relative to average levels throughout the day): insulin is high and glucagon and catecholamines are low.
Fuels in blood (relative to average): glucose is high, amino acids are high. Free fatty acids and ketone bodies are low.
Results: high levels of insulin direct all sensitive cells to take up, store and burn glucose. The liver and muscle make glycogen and the liver makes fatty acids from glucose (stored as TAG).
Phase II: 12 hours after meal - hormones, fuels and results
Hormone Levels: insulin low. glucagon and catecholamines are high.
Fuels in Blood: glucose is low. free fatty acids and ketone bodies are high.
Results: adipose hydrolyzing TAG to produce free fatty acid. free fatty acid uptake in liver to form ketone bodies. liver is hydrolyzing glycogen to make glucose. liver is running glucoeneogenesis.
Starved State (1 week after meal): hormones, fuels and results
Hormone Levels: insulin very low. glucagon and catecholamines are very high.
Fuels in Blood: glucose is very low. free fatty acids and ketone bodies are very high.
Results: Adipose continues to release free fatty acids to blood. liver uses free fatty acids to make ketone bodies. But brain still needs about 6g glucose/hour. The liver is out of glycogen. Only source of glucose is gluconeogenesis in liver. Carbon sources for gluconeogenesis are amino acids, lactic acid and glycerol (from breakdown of TAG).
Pathway Regulation
The rate of flux down a pathway is controlled by the activity and concentrations of the enzymes that catalyze the rate determining steps for that pathway. Two kinds: internal and external.
Internal Regulation
by metabolites of metabolic pathways. Short-term and focused on keeping the cell functioning. Example is ATP levels of a cell.
- feedback inhibition of rate limiting enzymes by products of pathway
- feedforward activation of rate limiting enzymes by substrates of the pathway.
- regulation by the relative concentrations of ATP/ADP/AMP.
External Regulation
by hormones via the stimulation of second messenger systems caused by binding of the hormones to receptors on the cell surface.
Control of glycolysis and gluconeogenesis in the liver by hormones
- Glycolysis is controlled by the enzyme PFK (phosphofructokinase). AMP and insulin (increases levels of F2,6-BP which is positive effector) are positive allosteric effectors. And have a decrease in F1,6-BP activity (responsible for gluconeogenesis).
- Gluconeogenesis is controlled by the enzyme F-1,6-BP. AMP is a negative allosteric inhibitor. F2,6-BP levels fall. High levels of the two hormones glucagon and epinephrine. And decrease in PFK activity.
Control of glycogenolysis in muscle and liver
- Glycogen synthesis: hormone is insulin. enzyme being regulated is glycogen synthetase (glycogen synthesis) and glycogen phosphorylase (glycogen breakdown). insulin dephosphorylates both enzymes, making the synthetase active and the phosphorylase inactive. Result is glycogen synthesis.
- Glycogen Breakdown: Hormones are glucagon and epinephrine. Both phosphorylate the enzymes making the glycogen synthetase enzyme inactive and the glycogen phosphorylase active. Result is glycogen breakdown.
- Note that liver has the glucose-6-phosphatase enzyme allowing glucose to be made. This means that the liver can produce glucose that can be exported to the blood but muscles cannot. Glycogen breakdown is for internal muscle use only.
- Calcium and ADP are positive allosteric effectors of glycogen phosphorylase
Glucogenic Liver
producing glucose via glycogenolysis and gluconeogenesis due to glucagon and epinephrine external regulation.
- glycogen undergoing glycogenolysis to glucose which is exported.
- Alanine and lactate act as carbon sources to form pyruvate and undergo gluconeogenesis to form glucose to be exported. glycerol is a positive allosteric effector of gluconeogenesis.
- Fatty acids from adipose undergo the TCA cycle to produce ATP and ketone bodies which are exported.
Lipogenic Liver
importing glucose to convert to glycogen, fatty acids/TAG exports. Controlled by insulin.
- glycolysis to form ATP from glucose.
- glycogen synthesis to form glycogen from glucose
- up regulation of lipids as VLDL
Effect of insulin on muscle
promoting glucose uptake, glycogen synthesis, burning of glucose. Also, low levels of free fatty acids in blood force muscle to burn glucose.
Effect of epinephrine on muscle with O2
glycogenolysis and glycolysis. large amounts of free fatty acids are taken up by muscle to form acetyl CoA. Starting to export lactate (produced from pyruvate) to send to the liver for gluconeogenesis. “Cori Cycle”
Effect of epinephrine and energy change on anaerobic muscle.
no TCA cycle (no oxygen). glycolysis is run to obtain ATP. lactate is exported to liver for gluconeogensis. Glycogenolysis is also run to produce glucose for glycolysis.
Review of Insulins effects on enzymes (five enzymes)
- glycogen synthetase activation: glycogen synthesis
- pyruvate kinase activation: glycogen synthesis
- acetyl CoA carboxylase activation: fatty acid biosynthesis
- F-2,6-BP activation: glycolysis
- glycogen phosphorylase inactivation: glycogen breakdown
Increasing Insulin: effect on glucose uptake
increases in liver adipose and muscle
Increasing Insulin: enzyme and effect on glycolysis
PFK via F2,6 BP. increases in liver, adipose and muscle
Increasing Insulin: Enzyme and effect on gluconeogenesis
F 1,6 BP. decreases in liver
Increasing Insulin: Enzyme and effect on glycogen synthesis
glycogen synthetase. increases in liver and muscle
Increasing Insulin: Enzyme and effect on glycogenolysis
glycogen phosphorylase. decreases in liver and muscle
Increasing Insulin: Enzyme and effect on fatty acid synthesis
acetyl CoA carboxylase. increases in liver
Increasing Insulin: Enzyme and effect on TAG uptake in adipose
LPL (lipoprotein lipase) and increases in adipose
Increasing Insulin: Enzyme and effect on TAG synthesis from fatty acids
glyerolphosphate acyle transferase. increases in liver and adipose.
Increasing Insulin: Enzyme and effect on lipolysis in adipose
hormone sensitive lipase (HLP). decreases in adipose
Increasing Insulin: Enzyme and effect on protein synthesis
induction of enzyme synthesis. increases in muscle.
Glucagon and Catecholamines
promote phosphorylation of regulatory enzymes via cAMP signaling cascades. Responsible for increasing the level of glucose, free fatty acids and ketone bodies in the blood.
Review of glucagon and catecholamine effects on enzymes (6)
- glycogen phosphorylase activation: glycogen breakdown
- enzyme that degrades F2,6BP activation: stops glycolysis
- glycogen synthetase inactivation: stops glycogen synthesis
- acetyl CoA carboxylase inactivation: stops fatty acid biosynthesis
- pyruvate kinase inactivation: stops glycogen synthesis
- promotes formation of ketone bodies from free fatty acids in the blood.
Review of glucagon effects on pathways (5 pathways)
remember only works in liver
- decreases glycolysis
- increases gluconeogenesis
- decreases glycogen synthesis
- increases glycogenolysis
- decreases fatty acid synthesis.
Review of glucocorticoids (chronic stress) effects on pathways (4)
- increases gluconeogenesis in liver
- increases glycogen synthesis in liver
- increases lipolysis in adipose
- increases proteolysis in muscle.
Increasing epinephrine effect on glycolysis
decreases in liver and adipose but increases in muscle
Increasing epinephrine effect on gluconeogenesis
increases in liver
Increasing epinephrine effect on glycogen synthesis
decreases in liver and muscle
Increasing epinephrine effect on glycogenolysis
increases in liver and muscle
Increasing epinephrine effect on fatty acid synthesis
decreases in liver
Increasing epinephrine effect on TAG uptake
decreases in adipose and increases in muscle
Increasing epinephrine effect on lipolysis
increases in adipose
Formation of Insulin
released from pancreatic beta cells. Brief steps: glucose enters cell, is burned to produce ATP which closes a potassium channel causing depolarization of the cell. A voltage gated calcium channel opens which promotes exocytosis of proinsulin. Proinsulin is cleaved to produce insulin and a peptide called C peptide.
Formation of glucagon
released from alpha cells in pancreas. Controlled by paracrine regulation. Glucagon is secreted when insulin levels fall and is inhibited when insulin levels rise.
Epinephrine and glycolysis in muscles
- catabolic hormone released from the adrenal gland into the general circulation. “fight or flight”
- activation of glycolysis in muscle via a secondary messenger: epinephrine binds to receptor on cell surface activating adenylyl cyclase which converts ATP to cAMP. cAMP is a positive allosteric effector of protein kinase A which phosphorylates and activates PF-2-K. PF-2-K catalyzes the conversion of fructose-6-phosphate to F2,6-BP which is a positive allosteric effector of the enzyme PFK important in glycolysis.
- note the pathway is different in the liver and in the liver epinephrine turns off glycolysis.
Diabetes Mellitus
characterized by inadequate amounts of insulin, inadequate response to insulin and high levels of blood glucose
Oral Glucose Tolerance Test
patient undergoes 10 hour fast followed by standard dose of glucose. Measure blood glucose at 30 minute increments up to 2 hours. levels under 140mg/dL are normal and over 200mg/dL are diabetic.
Type I DM symptoms, characteristics, metabolic pattern and cause
- symptoms: lethargy, weight loss, acetone breath, N/V, Kussmaul breathing (hyperventilation)
- Characteristics: juvenile onset, frequency is low and variable (no genetic component), lifespan is 70% of normal,
- Metabolic pattern: resembles starved state because only counter-insulin hormones are present. Also have high blood glucose and lipoprotein related triglycerides in blood. Observe fasting hyperglycemia in an attempt to preserve glucose.
- Cause: irreversible autoimmune destruction of pancreatic beta cells
HbA1C
glycosylated hemoglobin. observe high levels in diabetics. hemoglobin is irreversibly glycosylated through an Amadori arrangement, so it is a good measure of long term glycemic control. Above 6.5% is diabetic and levels between 5.7% - 6.4% are pre-diabetic.
Fructosamine test
measures the percent of plasma proteins that have been glycosylated. measures short-term glycemic control. Useful in gestational diabetes.
Treatment of Type I DM
insulin injections are only treatment option. Usually use two types: long acting to establish baseline levels and short acting for postprandial glucose uptake.
long acting insulin: insulin glargine and detemir insulin
short acting insulin: insulin lispro and insulin aspart
Negative outcomes of Type I DM
hyperglycemia, glucosuria (glucose in urine) and polyuria. Diabetic ketoacidosis (high levels of ketone bodies in blood) which may lead to coma and death.
Hypoglycemic Shock
occurs in insulin overdose.
symptoms: uneasiness, trembling, irritability, cold sweats. It is difficult for an experienced diabetic to recognize early signs.
Treatment: depends on severity. candy and fruit juice for mild. Or IV glucose and potassium for intermediate. Severe is glucagon injections.
Characteristics: increased efforts for high levels of control of diabetes results in a 3-fold increase in incidence of hypoglycemic shock.
Type II DM symptoms, characteristics, metabolic patterns
symptoms: blurred vision, polyuria, polydipsia (thirsty), polyphagia (interested in eating
Characteristics: obesity, adult onset, reduced lifespan due to toxic effects of glucose.
Metabolic Pattern: fasting glucose higher and response to glucose after meal is exaggerated.
Type II DM causes
insulin deficiency and/or decreased response (insulin resistance or decreased insulin receptors). Insulin resistance is a component of metabolic syndrome. Underproduction of insulin by pancreas increases severity.
Type II DM negative outcomes
nonketotic hyperosmolar coma due to high glucose levels. diabetic ketoacidosis is rare.
Type II DM treatment (6 drug classes)
- in general a class of drugs will lower HbA1C by 0.7-1%
- diet always first. weight reduction reduces type II DM in 50% of patients who can maintain normal body weight.
- oral sulfonylureas: increase insulin secretion from pancrease. tolbutamide and glipizide
- thiazolidinedione agents: increase sensitivity of insulin receptors. pioglitazone. heart problems
- Metformin: inhibits gluconeogenesis. first line drug.
- GLP-1 agonists: stimulates insulin release and suppresses glucagon release. Byeatta.
- DPP-4 inhibitor: DPP-4 degrades GLP-1. Januva
- SGLT-2 inhibitors: inhibits reabsorption of glucose back into kidney. increases glucose spilling. Invokana, Canagliflozin
Hyperglycemic Toxic Effects
- vascular disease: thickened blood vessel walls, poorer circulation, coronary heart disease, may lead to ulceration and amputation of foot. AGE related
- kidney malfunctions
- retinal degeneration and cataracts (maybe brought on by AGE)
- neuropathy: nerve damage due to glycosylation of proteins through Amadori rearrangements. Irreversible. Protein crosslinking eventually leads to advanced glycation end products (AGE)
Replication
synthesis of new DNA using the existing DNA as a template
Transcription
synthesis of RNA using a gene sequence (DNA) as a template
Translation
synthesis of a protein using an mRNA transcript as a template
DNA polymerases
enzyme that catalyzes addition of deoxynucleoside monophosphates to DNA strand. Catalyzes DNA replication.
nucleophilic attack by 3’ hydroxy group of a primer strand on the incoming dNTP.
transesterification reaction
5’ to 3’
driving force is hydrolysis of PPi by inorganic pyrophosphatase
Requirements for DNA polymerases
- template strand
- primer strand with a free 3’ OH
- dNTPs
- Magnesium
Pol I
Prokaryotic DNA polymerase.
Has three main functions. Not essential in DNA replication but more important in repair.
- DNA polymerase activity. moderate processivity (20bp/encounter and moderate fidelity (10^-5 errors/bp)
- Exonuclease Activity 1: chews up DNA from the 3’ –> 5’. Boosts fidelity to 10^-8 errors/bp
- Exonuclease Activity 2: chews up DNA from 5’ –> 3’.
Pol III
Prokaryotic Polymerase. replication of DNA. high processivity (1000bp/encounter), very fast (1000bp/sec). moderate fidelity (10^-5 errors/bp). Has exonuclease 1 activity (3’–>5’ proofreading) which boosts fidelity to 10^-8 errors/bp.
DNA replication in E. coli: Initiation Phase (7 steps)
- dnaA protein binds to oriC which results in unwinding of the DNA
- dnaB and dnaC further unwind
- SSB (single stranded binding protein) binds to and stabilizes unwound DNA.
- bubble forms at oriC with unwinding in DNA occurring in both directions.
- Primase (RNA polymerase) binds to single-stranded DNA in a multiprotein complex known as a primosome.
- Primase synthesizes short RNA primers
- Primase dissociates
DNA replication in E. coli: Elongation Phase (4 steps)
- Replication fork is formed when Pol III assembles onto DNA.
- Leading strand is the 3’ to 5’ so replication by pol III occurs in the 5’ to 3’ direction
- Lagging strand is the 5’ to 3’ so replication occurs in Okazaki fragments and ligase joins the fragments together.
- DNA gyrase introduces negative supercoils into the DNA to prevent supercoiling caused by unwinding of the DNA.
DNA replication in E. coli: Termination Phase (2 steps)
- replication ends when the replication forks meet at the termination region (ter).
- topoisomerase IV is responsible for resolving the interlinking of the “concatanes”. Two circles of DNA will be stuck inside one another.
Fluoroquinolones
Antibiotics that inhibit DNA gyrase and topo IV enzymes.
DNA mismatch repain
In DNA replication. MutS and MutL recognize incorrect pairing because of methylation of parent strand. They chop up daughter strand for repair by a DNA polymerase.
DNA damage repair
usually caused by UV light, X-rays, reactive oxygen species etc…
DNA glycosylase removes the damaged base to create an AP site. Then AP endonuclease chews up the backbone in that area Pol I and DNA ligase repair the site.
For nucleotide excision repair Uvr-A or B or C removes the backbone and Pol I and DNA ligase repair.
Bacterial Plasmids
circular, extra-chromosomal, double stranded DNA molecules that replicate autonomously. May be transferred between bacteria and often carry genes responsible for antibiotic resistance and toxins.
DNA replication in eukaryotes
similar to in prokaryotes
- DNA polymerase a : primer complex synthesizes an RNA primer and then adds about 15 bases of DNA.
- The primer is removed by FEN-1 nuclease
- DNA pols ∂ and epsilon are the main polymerases involved in replication
- other polymerases function mainly in repair pathways. examples are pol ß and pol gamma is found in mitochondria and chloroplasts.
Camptothecin and Etoposide
Topoisomerase responsible for unwinding DNA during replication. Camptothecin and Etoposide are topoisomerase poisons that cause the DNA to break while replicating. They are used in cancer chemotherapy.
RNAP
RNA polymerase which is the enzyme responsible for transcribing DNA. Require template strand, rNTP (ribonucleoside triphosphate) and magnesium (no primer necessary)
RNA Core enzyme - no sigma subunit
RNA Holoenzyme - has the sigma subunit
Gene
segment of DNA that is transcribed for the purpose of expressing the encoded genetic information as a protein.
Promoter
where transcription begins. contains a consensus sequence that controls the rate of initiation. “Strong” promoters more closely match the consensus sequence and initiate transcription much quicker than “weak” promoters who differ in the consensus sequence.
Transcription Initiation (4 steps)
- RNAP Core enzyme highly processive but cannot initiate RNA synthesis. RNAP holoenzyme binds to DNA and the sigma subunit recognizes the promoter sequence.
- Sigma subunit stops at the promoter sequence and forms the closed promoter complex.
- RNAP then unwinds DNA to form the open promoter complex
- About 8-10 nucleotides are added and the sigma subunit dissociates from the holoenzyme. This process is called promoter clearance.
Transcription Elongation
- RNAP core enzyme highly processive and RNA synthesis continues until the entire gene has been transcribed.
- transcription bubble: region containing the RNAP core enzyme, DNA and the RNA transcript. only have 1 fork.
- synthesis happens at about 40bp/sec
- fidelity of RNAP is 10^-4 to 10^-5 errors/bp and lacks any editing function.
- end of the gene contains stop signals: either row-dependent or row-independent.
Promoter sequences and sigma factors in transcription regulation in prokaryotes
Each sigma factor recognizes a specific promoter sequence. 70 is houskeeping. 38 is starvation/stationary phase. 32 is heat shock. 24 is extracytoplasmic stress.
sigma 32 for heat shock: only activated at elevated temperatures.
Three methods of transcription regulation in prokaryotes
- promoter sequences and sigma factors.
- inducers and repressors. lac operon example.
- Catabolite repression. glucose example
Inducers and Repressors in Transcription Regulation
- Lac operon. transcription produces three genes required for lactose utilization in E. coli
- lac repressor is a protein that binds to the operator site (O) and prevents RNAP from binding to the promoter site (P).
- lac repressor is a negative regulator of transcription.
- When lactose is present in the cell it is metabolized to allolactose which binds to lac repressor. This complex dissociates from the DNA which allows RNAP to bind to the promoter region of the lac operon and stimulate transcription.
- allolactose is an inducer.
Catabolite Repression in transcription regulation
- When glucose is present in the cell, want to turn off genes for proteins involved in metabolism of other catabolites like lactose etc…
- glucose in the cell inhibits the enzyme adenylate cyclase which produces cAMP
- cAMP serves as a “hunger signal” and binds to Catabolite Gene Activator Protein (CAP) which enhances its affinity for DNA.
- The cAMP : CAP complex binds to the promoter region of the lac operon (among others) and stimulates transcription. It is a positive regulator.
Inhibitors of Prokaryotic Transcription
rifamycin B and its semi-synthetic derivative rifampicin inhibit transcription.
RNAP in eukaryotes
Three RNAP enzymes: Type I (rRNA), Type II (responsible for mRNA transcription) and Type III (tRNA)
Initiation Transcription in Eukaryotes
- No sigma factors to find promoter, instead they have transcription factors that bind to promoter and recruit RNAP II.
- General transcription factors assemble at the TATA box to form a pre-initiation complex.
RNAP II initiates RNA synthesis and departs from promoter leaving transcription factors behind.
Death Cap Mushroom
contains toxin called alpha-amanitin which is an inhibitor of eukaryotic RNAP II inhibiting the elongation phase of RNA synthesis.
Post-transcriptional mRNA processing
eukaryotes only. (in nucleus)
5’-capping: modified G attached to the 5’ end of the transcript through a 5’ –> 5’ bond.
3’-polyadenylation: string of A’s added to the 3’ end of the transcript.
splicing: introns are removed. remaining exons are joined together. Alternative splicing can generate multiple distinct mature mRNA’s from a single gene. Splicing is carried out by the spliceosome.
Regulation Transcription in Eukaryotes. Silencer:Repressor and Enhancer:Activator
activators bind to enhancers elements and aid in RNAP recruitment to the promoter.
silencer binds to repressor and downregulates transcription (i.e. is lac repressor protein)
Regulation of Transcription in Eukaryotes. Chromatin Remodeling.
- fundamental unit of chromatin is a nucleosome. Each nucleosome core particle consists of about 146 bp of DNA wrapped around an octamer of histone proteins
- polysomes: linking of nucleosomes so that they look like beads on a string.
- HAT: histone acetyltransferases add acetyl groups to histone Lys residues leading to transcriptional activation.
- HDAC: histone deacetylases reomve acetyl groups from histones and are associated with gene silencing
- H1 interacts with inter-nucleosome linker segments. histone proteins are H2A, H2B, H3 and H4
Regulation Transcription in Eukaryotes: DNA methylation
CpG islands of sites of methylation causes decreased gene expression. methylcyosine binding protein further acts as a transcriptional repressor. Associated with epigenetic inheritance.
Regulation Transcription in Eukaryotes: miRNA processing
- miRNA regulate gene expression through RNA interference
- primary pri-miRNA is cleaved by drosha into pre-miRNA and exported from the nucleus.
- Dicer cleaves pre-miRNA into siRNA.
- RISC binds siRNA and guide strand forming a complex that blocks translation and stimulates degredation of the polyA tail.
Point mutations.
DNA translation. changing a single nucleotide in the sequence.
- silent mutation: point mutation that codes for the same amino acid
- missense mutation: codes for a different amino acid
- nonsense: turns an amino acid into a stop codon
- read-through: turns a stop codon into an amino acid codon.
Frameshift Mutation
DNA translation. addition or deletion of a single base in the mRNA transcript.
Amide Bond formation.
DNA translation. energetically unfavorable condensation reaction. energy barrier is overcome by activating the amino acid to an aminoacyl-tRNA.
Aminoacyl-tRNA synthesis.
DNA translation. aminoacyl-tRNA synthetases
- responsible for activating amino acids.
- driven by inorganic pyrophosphatate.
- two high energy bonds are hydrolyzed to activate each amino acid.
Initiation of Translation in Prokaryotes.
- Shine-Delgarno Sequence is the initiation codon in prokaryotes. Anti-codon of tRNAfmet recognizes an AUG codon
- Assembly of initiation complex which includes initiation factors (IF1, IF2, IF3).
- polycistronic genes: mRNA with multiple Shine-Delgarno sequences so multiple proteins are being translated at the same time. Only in prokaryotes.
- Skip the A site and go straight to the P site
- GTP hydrolysis by IF2 is required to assemble the functional ribosome.
Elongation of Translation in Prokaryotes
- 2EF-Tu delivers an activated tRNA to the A-site. If properly paired EF-Tu hydrolyzes GTP and dissociates from the ribosome. Note this is a GTP hydrolysis proofreading step.
- peptidyl transferase of the 50S ribosomal unit catalyzes trans-peptidation reaction.
- Nascent chain grows in the N –> C direction.
- Translocation slides the ribosome one codon toward the 3’ end putting the peptidyl-tRNA in the P site and an uncharged tRNA into the E site. This process is powered by GTP hydrolysis.
Termination in Translation in Prokaryotes
- Stop codons are recognized by release factors which bind to the A-site and trigger hydrolysis of the protein from the tRNA leading to dissociation of the ribosome.
- Requires one GTP.
Translation Differences in Eukaryotes vs. Prokaryotes.
ribosome bigger
no Shine-Delgarno sequence. The 5’ cap is responsible for initiation of protein synthesis.
Monocistronic: one gene, one protein.
Name the Five Inhibitors of Translation and their Mechanism
- Aminoglycosides: inhibit fmet-tRNA binding and impair proofreading
- Tetracyclines: inhibit aminoacyl-tRNA binding to the A site
- Chloramphenicol: inhibits peptidyltransferase activity
- Macrolides: inhibit translocation to P-site
- Puromycin: mimics 3’ end of aminoacyl-tRNA causing premature termination in prokaryotes and eukaryotes therefore is not used clinically only in lab.
Protein Structure
Primary structure: linear amino acid sequence
Secondary structure: local, regular arrangement of amino acids stabilized by hydrogen bonds. Alpha helix and Beta sheets.
Tertiary structure: compact 3D structure of a single polypeptide chain
Quaternary structure: arrangement of multiple polypeptide molecules in a multi-subunit complex. Picture is hemoglobin.
Amino Acids with Positive Side chains
Arg, His, Lys
Amino Acids with Negative Side Chains
Asp, Glu
Amino Acids with polar uncharged side chains
Ser, Thr, Asn, Gln
Special Cases Amino Acids
Cys, Gly, Pro
Amino Acids with hydrophobic side chains
Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp (AVILMPTT)
Forces that drive protein folding
hydrogen bonding, charge-charge interactions, hydrophobic effect, vanderWaals forces.
Holdases
- bind misfolded or incompletely folded proteins until they can spontaneously assume their correct fold
- Ubiquitous holdases: heat shock proteins. HSP27 and aB-crystallin (abundant in the eye). bind to unfolded/misfolded proteins in cell to prevent their aggregation
Hsp70
Foldases.
DnaK (prokaryotes) and Hsc70 & Hsp70 (eukaryotes).
resting state is ATP bound.
Cochaperones DnaJ or Hsp40 facilitate binding of misfolded client proteins.
ATP hydrolysis by NEF (nucleotide exchange factor).
GroEL and Hsp60s
- GroEL is the barrel and has hydrophobic patches and nucleotide binding sites. GroEL is ATP bound
- Caps are GroES traps client in cis chamber and blocks hydrophobic site which promotes folding.
- GroES causes hydrolysis of ATP in cis chamber.
- Once folded, binding in the trans chamber of a new client protein displaces cis ADPs and and cis GroES.
- symmetrical process on trans side.
Tanespimycin
Tanespimycin and other Hsp90 inhibitors are currently being developed for cancer therapies. Hsp90 is over-expressed in many forms of cancer.
Ubiquitination System
- Ubiquitin is attached to Ub-activating enzyme (E1) through consumption of ATP.
- Ubiquitin is transferred to Ub-conjugating enzyme (E2)
- E2 and ubiquitin complex binds E3 and substrate complex.
- Ub is transferred to a lys residue on the substrate
- Process is repeated to get polyubiquitination
Proteasome
- Regulatory (19S) particles: recognition of polyubiquitinated substrates (requires ATP binding), deubiquitination and release of Ub, substrate unfolding (ATP hydrolysis), translocation to core particle
- Core particle (20S): site of proteolysis.
Bortezomib
approved for treatment of relapsed multiple myeloma. Inhibits proteasome.
Amyloid
insoluble, B-sheet protein aggregates. generally display “templated” or “seeded” aggregation in that a misfolded protein can induce the conversion of native protein into a misfolded form.
Prion Diseases
templated protein misfolding. PrP acts as infectious particle. Example is Mad Cow Disease.
Huntigton’s Disease
genetic disorder characterized by aggregation of huntingtin protein. gene for huntingtin has a variable number of CAG repeats that encode for a stretch of Gln. The number of CAG repeats accurately predicts age of onset (higher number is earlier onset).
Familial Amyloid polyneuropathy and Senile systemic amyloidosis
caused by aggregation of transthyretin (TTR) and requires the dissociation of the native TTR tetramer into monomers. Drug Tafamidis binds to and stabilizes the tetramer potently inhibiting aggregation and arresting disease progression.
Cystic Fibrosis
mutations in CFTR ion channel that lead to degradation of protein or misfolding. Ivacaftor can bind to misfolded CFTR and force it into its native and functional state.
Posttranslational Modification and Collagen
triple-helix structure. Gly-Pro-Pro repeats. Three types of posttranslational modifications.
- Pro residues hydroxylated to hydroxyproline, enhancing the stability of the triple helix
- Lys residues hydroxylated to hydroxylysine and then O-glycosylated, important for signal functioning
- Inter-strand disulfide bonds between Cys trigger formation of triple helix.
Protein Targeting and Ribosomes
Ribosomes in the cytosol: synthesize proteins destined for the cytosol, nucleus, mitochondria
Ribosomes bound to the ER: synthesize proteins bound for the ER, Golgi apparatus, lysosome, plasma membrane or the extracellular environment.
Mitochondrial Protein Targeting
- mitochondrial matrix targeting sequence targets the protein to the mitochondria.
- TOM and TIM (translocase of outer/inner membrane) move preprotein through the mitochondrial membranes where Hsp70 and Hsp60 are standing by.
- MPP cleaves off the target sequence once inside the mitochondria.
Nuclear Protein Targeting
- Nuclear Pore complex: proteins smaller than 15 kDa can spontaneously enter.
- Larger proteins are targeted by a nuclear localization sequence which are recognized by importins which facilitate transport through the nuclear pore complex.
Protein Targeting from the ER
- signal peptide shows where protein is destined for. Recognized by signal recognition particle (SRP) as they emerge from the ribosome. When SRP binds protein synthesis halts.
- SRP-ribosome complex diffuses to the ER surface where it binds the SRP receptor and translocon complex.
- GTP hydrolysis triggers dislocation of SRP from ribosome-translocon complex.
- Translation continues. Signal peptidase cleaves the signal peptide from growing strand. If the signal peptide is not removed the strand is called preprotein
- When synthesis is complete it is released into the ER and the ribosome dissociates.
Membrane Protein Targeting
Same process as protein targeting from the ER but instead have start transfer signals and stop transfer signals
Start transfer signals: NH3 side of protein is in cytosol and COO- is in the lumen of the ER.
Stop transfer signals: COO- in cytosol and NH3 in ER lumen.
N-linked Glycosylation
- added while the protein is in the ER lumen (core glycosylation).
- Lipid cofactor of dolichol phosphate forms the core oligosaccharide which is attached.
- Glycosidases trim certain monosaccharides.
- Tunicamycin is an antibiotic that inhibits the addition of monosaccharide units to dolichol.
O-linked glycosylation
Ser and Thr. Added in the Golgi Apparatus. Added one monosaccharide at a time. proteoglycans are proteins with 100s of disaccharide units.
Bacterial Wall Biosynthesis
- cross-linked peptidoglycans
- bactoprenol is the equivalent of dolichol (responsible for the core oligosaccharide in N-glycosylation).
- Bacitracin inhibits enzyme responisble for dephoshporylation of bactoprenol.
- Beta-lactams inhibit transpeptidase, the enzyme responsible for cross-linking the peptidoglycan chains.
