BIO 1A03 - Test 2

What are aminoacyl tRNA synthetases? what do they do?

They are a family for enzymes that match/attach the correct amino acid onto its tRNA. They do so by using their active site to recognize the anticodon end of tRNA molecules after they bind to it, and catalyze a covalent bond between tRNA and amino using energy from ATP hydrolysis. They are unique to the type of tRNA and amino that they are meant for (There are 20, for 20 amino acids)

What are the 5 cellular components required for translation? Small description of their functions?

1. Elongation and release factor proteins (bring the tRNA to the ribosome, and recognize a stop codon to terminate transcription)
2. tRNA (binds to amino acids, and then binds to mRNA through a peptide bond and transfers its amino acid/chain onto the newest added amino acid)
3. Synthetase enzymes (matches the tRNA to its correct amino acid)
4. rNA
5. ribosomal proteins (makes up the ribosome which is the core machinery where tRNA works in)

Where does tRNA obtain its amino acids from?

The cytoplasm of the cell

What are tRNA molecules made of? What is the structure like?

A single RNA strand that is 70-90 nucleotides long. This forms 4 double helical segments, and 3 characteristic loops which gives its “clover shape” appearance. The last double helical segment is open, and is the amino acid attachment site. The whole molecule folds into a 3D L-shaped structure

Where do amino acids bind on the tRNA molcule

At the open-ended double helical-structure, on the protruding 3’ end (there are 3 codons open)

What is tRNA molecule activation?

The covalent bonding of an amino acid to the tRNA attachment terminal

What is the anticodon region?

The nucleotide triplet on the bottom loop on a tRNA molecule. This base pairs complementary to a specific mRNA codon, to code for a specific amino acid

What direction is the anticodon region written in?

3’ to 5’, since it pairs with an mRNA that is written 5’ to 3’

T or F, there are 20 tRNA molecules for the 20 amino acids

F, there are 45 tRNA molecules and some tRNA anticodons can bind to more than one mRNA codon

What is the wobble hypothesis? What does this explain?

The 3rd position in the mRNA codon, and the 1st position on the tRNA anticodon can undergo improper base pairing. This explains why multiple codons can code for a single amino acid, and why there are more codons than tRNA molecules (tRNA can pair with more than 1 codon)

In simple terms, how do eukaryotic and prokaryotic transcription initiation differ?

Eukaryotes: Initiation complex forms towards the 5’ cap on mRNA, and the scans the strand until AUG is found

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Prokaryotes: No 5’ cap. Initiation complex assembles at (1 or more) Shine-Dalgarno sequences, which are binding sites located a few bases upstream of AUG

Polycristonic

mRNA that codes for multiple proteins (single strand, only in prokaryotes)

What is a unique translation ability that prokaryotes have?

They can simultaneously translate seperate proteins along a single strand of mRNA. This can occur because their genes of similar functions are grouped along their DNA, which are often transcribed as a single unit from one promoter

T or F, the ribosomal subunits will only assemble after the small subunit attaches to the RNA

T, for both eukaryotes and prokaryotes

What are the 4 components required for translation initiation?

1. 2 ribosomal subunits (large and small)
2. mRNA that requires translation
3. Charged tRNA methionine (tRNA that’s bound to an amino acid)
4. Initiation factors that assist with initiation complex assembly

What is each section of the ribosome?

Left: E-site (where tRNA exits)

Middle: P-site (peptidyl site, where methionine is the first to join)

Right: A-site (aminoacyl site, where incoming tRNA enters the ribosome)

What causes deacylated tRNA to exit the ribosome?

Energy from GTP allows the ribosomes continue to translocate along the length of the mRNA molecule, and new tRNA molecules enter the ribosome as new codons are recognized. This shifts the previous tRNA to the next position in the ribosome, and when one shifts from P to E site, it can be released

T or F, the binding of tRNA at the A-site requires energy

T, it requires GTP

Neurospora crassa

Type of bread mold

Experiments done in the 1940’s established a relationship between genes and proteins

Why can neurospora crassa grow on a medium lacking amino acid arginine?

They are able to synthesize their own, happens by:

Precursor → Ornithine → Citrulline → Arginine

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This metabolic pathway requires a specific enzyme to get to the next step

One-gene-one-enzyme hypothesis

How does neurospora support this?

Theory that each gene directly produces a single enzyme, which will affect the metabolic pathway it is involved in

* Neurospora can grow well on minimal medium (contains simple substances)
* Must produce some enzymes (from a specific gene) that convert simple substances into amino acids/vitamins needed for growth

How did G. Beadle and E. Tatum set up their research?

Performed a genetic screen of radiation treated Neurospora, (they knew radiation would lead to potential mutations) and raised colonies of radiation treated cells on a medium either supplemented with nothing, or with only one of the intermediates added 

What were G. Beadle and E. Tatum’s findings in their research? (4 components)

Arginine supplemented medium: continual growth

Non-supplemented medium: no growth

Single intermediate medium: inhibition of growth

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They assumed that radiation produced mutations in the genes encoding necessary enzymes for arginine production

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They identified that genes code for enzymes AND that genes dictate the structures of all proteins, turning the one-gene-one-enzyme hypothesis into the one-gene-one-polypeptide hypothesis, because not all proteins are enzymes



Ornithine is a precursor to citrulline, and citrulline is a precursor to arginine

What are the exceptions to the one-gene-one-polypeptide hypothesis

(think what the hypothesis means!)

1. There are 20-25,000 protein-encoding genes identified in the human genome. (evidence that more than one protein can possibly be produced from a single gene) 
2. Alternative splicing of genes contribute to the diverse number of mRNA transcripts, meaning there is added complexity that exists from genome to proteome (post translational modifications of proteins translated from the same DNA allows for the production of diverse proteins) 

Human Proteome

Represents the full number of proteins expressed by all hereditary information in our DNA (our genome), composition can change due to various factors, such as developmental stage

What happens when your blood glucose levels rise after a meal? What is this an example of?

The pancreas will modulate synthesis and secretion of its signal (the protein insulin)

This is a cellular sensory response

Insulin

Effector protein produced by pancreatic beta cells. These proteins communicate with target cells, to create a response to decrease blood glucose levels, allowing glucose to enter cells

* Its polypeptide is 110 amino acids, but final protein is only 51 amino acids

How did Dorothy Hodgkin determine the structure of Insulin?

Using X-ray crystallography techniques

What is the structure of the insulin protein?

A dimer (bonded monomers, which are parts of polymers)

* Made of 2 amino acid chains
* Alpha chain + beta chain

Where does glucose absorption occur from eating?

* Some occurs in mouth across epithelial surfaces, associated with underlying blood vessels/capillaries
* Large amount occurs in microvilli cells in small intestine

Microvilli cells

Cells in the small intestine, that are intimately associated with blood vessels which absorb glucose and transfer it to the bloodstream

Pancreatic Beta cells

Detector cells, that recognize an increase in blood glucose levels and responsively adjust the synthesis and secretion of insulin

What is preproinsulin

Precursor of the mature insulin protein

* Translated by bound ribosomes, and then processed within the ER
* Contains a signal sequence that interacts with SRP to help transport preproinsulin into the ER lumen

What is Proinsulin

Cleaved version of proinsulin (signal sequence lost, sulfates are bonded)

* Modified by post-translational modifications
* Folds by the help of chaperone proteins, and forms 3 disulfide bonds
* Transported from rough ER to GA where further cleavage occurs to produce the mature dimer

What is an SRP

Signal Recognition Particles

Why are post translational modifications of insulin proteins so important?

The N-terminal (end) and C-terminal (internal end) amino acid residues in the A and B chains are ONLY able to bind to insulin receptors on target cells IF the post-translational modifications occur

What are examples of covalent attachments? Why are they important?

1. Phosphorylation (kinase enzymes covalently attach phosphate groups)
2. Methylation (covalent attachment of a methyl group)
3. Acetylation (covalent attachment of an acetyl group)

Important because they increase functional diversity of the proteome

what do kinase enzymes do?

Phosphorylate proteins/other molecules

What does insulin bind to? What does this cause

Receptor kinases, allows cells transport glucose across the plasma membrane into cytosol

What are the steps of glucose absorption

1. Extracellular insulin signal
2. Series of cytoplasmic proteins become sequentially activated
3. Intracellular response
4. Activation of glucose transporter proteins at cell surface
5. Glucose can be absorbed

What does a neg-neg feedback loop do?

Double negative: inhibitor is inhibited

How is excess glucose stored?

as triglycerides (fats)

T or F, Liver cells are better at absorbing glucose than muscle cells (what causes this?)

F, For skeletal muscles, exon 11 is removed from the mature mRNA product, producing a higher affinity version of the insulin receptor (these cells will now be able to mount a higher response of glucose uptake and meet their high energy needs)

Exon 11 is retained in liver cells

Alternative splicing

Primary transcripts from the same gene are spliced at different junctions to result in many different mature mRNA molecules, that each contain their own combinations of exons (helps with gene regulation)

T or F, Some exons can even be excluded during splicing

T, because it is possible for them to be identified as introns in some primary transcripts 

Isoform

different types of mature mRNA

Process of regulating glucose levels in blood 

1. Glucose levels rise, and are detected by sensor cells of the pancreas 
2. Insulin acts as an effector signal that targets cells of the body to absorb glucose from the bloodstream 
3. The signal to increase glucose absorption is regulated 
4. Once glucose levels are returned to resting levels, there is feedback to bring the system back to resting point (negative feedback loop) 
5. The feedback limits any further response in the entire system 

How do antibiotics work?

They work through translation, by disrupting translation at the site of the ribosome

What does amplification do in gene signaling?

Makes the process easier and more efficient

What is byssus? why is it important?

A waterproof adhesive created by marine mussels that help them cling to rocks. Can be genetically engineered by inserting mussel DNA into yeast cells, which translate the genes into byssus which has potential to be surgical glue

What allows byssus to adhere?

Knobby coating, protein fibers

Why are mussels losing their grip lately?

Climate change. Higher temp → lower oxygen levels

* These conditions weaken byssal fibers

How is the kermode bear a thing?

A single nucleotide mutation results in a modified protein product from the mc1r gene, responsible for regulating skin and hair colour (T → C replacement at codon 298)

What is significant about Eden Atwood

She lives with androgen insensitivity. A mutation in a single gene causes androgen (testosterone) receptor proteins to be faulty and unable to respond to testosterone

* She has XY chromosome but looks like a biological female

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Which of the following would be least likely to be implemented during translation?

a) release factors

b) Initiation factors

c) Elongation factors

d) sigma factors

e) A and B only

d) sigma factors, because they are only in bacteria, and associated with initiation

What 4 things are important for cell growth?

1. amino acids
2. vitamins
3. nucleotides
4. carbohydrates

Housekeeping genes

Genes in the DNA that is required AT ALL TIMES for normal function

* Always expressed (being transcribed and translated)
* Usually code for ribosomal proteins, or structural
* Allows for constant general cellular activities

What happens to E.coli cells when glucose sources are used up

Bacterial growth is arrested, but they can metabolize and alternate fuel source. They use up all glucose, then there is a period of no growth (while B-Galactosidase genes are turned on), and then the cell growth continues using lactose

What signals the switch between glucose and lactose usage in e.coli cells?

products of glucose metabolism, B-galactosidase is only transcribed if lactose is present in the absence of glucose

Why is glucose easier to metabolize in e.coli?

Because its a monosaccharide, and lactose is a disaccharide which must be cleaved at the beta glycosidic linkage into glucose and galactose by B-galactosidase

What did Francois Jacob and Jacques Monod observe when they grew e.coli on a lactose free-medium, while adding and taking away glucose?

Levels of B-galactosidase began to steadily increase in response to addition of lactose, and production ceased once lactose was removed

What 3 components of gene expression must be completed for protein coding genes? Which of the 3 are the slowest? Fastest?

Transcriptional control (SLOWEST, but most energy efficient)

Translational control

Post translational control, modifications (FASTEST)

T or F, variation in expression must depend on all 3 of the following:

Transcriptional control

Translational control

Post translational control

F, it may only depend on one

What is tRNA molecule activation? (as a step)

It is step 1: Ability of RNA polymerase to bind or transcribe a gene

* Activation requires that proteins bind to a promoter and increase binding of RNA polymerase 
* By controlling the binding of proteins to the promoter, the cell can either activate or inhibit transcription
* Cell starts from scratch, so its the slowest step

Translational regulation

Step 2: Translation rates, lifetime of mRNA

* controls the amount of proteins made from mRNA,  


* The rate translation occurs at, will determine the amount of protein produced, and the amount produced depends on the stability of the mRNA (if it is quickly degraded, little protein will be made) 

Post-Translational Regulation

Step 3: Modifications that change folding/protein activity

* allows the cell to have a stockpile of protein in the cell that is inactive 
* Once the cell receives the appropriate signal, it can lead to a simple modification to turn on all inactive proteins  

What level is production of lactose metabolizing enzymes regulated

In prokaryotes, the control in enzyme production is regulated at the transcriptional level

Lactase permease

Function? Location?

Transport protein sitting in the bacterial cell membrane, which allows the transport of lactose into the bacterial cells  

Beta-Galactosidase

Function? Location?

Enzyme, that’s cytoplasmically situated inside bacterial cells that cleaves the imported lactose into glucose and galactose

What are operators? What are they meant to do? Are they regulated?

Operators are sequences of nucleotides near start of operon (on/off switches), which control gene clusters on polycistronic mRNA, which are regulated to either allow or inhibit transcription

What are the components of an operon?

* a promoter 


* An operator (sequence of nucleotides near start of operon, on/off switch) 
* Coordinated gene cluster (whose products function in a common pathway)

What happens when the operator is not bound to any transcriptional inhibitor?

The RNA polymerase is able to attach to the promoter and transcribe 

What is the lac operon

Transcriptional regulator in prokaryotes, controls expression of the lactose metabolizing enzymes 

* Operator (lacO) is the binding site for a repressor protein, which is expressed by the lacI coding sequence

What are the 2 GENES that code for the necessary lactase metabolism proteins? What does each one code for?

1. LacY gene (codes for lactose permease)
2. LacZ gene (codes for B-galactosidase)

RNA polymerase can simply bind and activate transcription

T or F, RNA polymerase can freely transcribe the lac genes

F, lacI gene controls the expression of them

How are lactase metabolizing proteins negatively transcriptionally regulated?

LacI gene (codes for the repressor protein, which binds to the operator and inhibits transcription)

* Repressor protein is continuously expressed at low levels (usually when glucose is present) 

Where does the repressor protein bind?

to the operator region of the operon, and turns off transcription (since RNA polymerase is no longer able to bind to the promoter region)

What is the structure of the repressor protein

The repressor is a tetrameric protein made of 4 identical protein subunits, which binds tightly to the operator regions of the lac operon DNA 

* When all 4 subunits bind to the operon, the DNA is twisted into a loop 

Why is lactose known as an inducer molecule?

By binding to the repressor proteins on the lac operator (which causes a conformational change in the repressor so it can no longer blocks DNA), which induces transcription

What is cAMP?

Cyclic AMP, a secondary messenger made from ATP adenylyl cyclase

* Gives RNA polymerase the ability to bind to the lac promoter


* Increases in cAMP levels will contribute to the positive regulation of the lac operon
* Concentration of cAMP is an indicator of the general nutritional state of E.coli cells (and other bacteria cells)

What does high glucose levels do to cAMP levels? Why?

High glucose levels surrounding a cell will cause inhibition of adenylyl cyclase (enzyme) which prevents catalysis of cAMP production (meaning glucose up = cAMP down) 

Low glucose levels will cause bacterial cells to accumulate high levels of cAMP due to increased activity of adenylyl cyclase 

What proteins are involved in the positive regulation of the lac operon?

* cAMP
* CRP (Catabolite Activator Protein, activates transcription)

How does cAMP positively regulate transcription?

* When bound to CRP, it will bind to a different site on the bacterial DNA called the CRP-cAMP or CAP-cAMP binding site 
* Concentration of cAMP will determine whether a transcriptional activator will bind to this site (and also the degree of positive regulation) 
* cAMP binding to CRP(transcriptional activator protein) is an allosteric activator (binds away from active site to change conformity in order to make it easier to bind to active site)
* Transcription of B-galactosidase and lactase permease is activated

What does it mean if:

Transcription in ON

Transcription is OFF

ON: operator on, RNA polymerase can bind to a promoter sequence 

OFF: repressor protein binds to operator region

What is an inducer protein?

* Can bind to repressor and prevent them from binding to the DNA 
* Can cause negative transcriptional regulation to be allosterically inhibited (conformity change)


* Inducers are inducers of genes

T or F : Glucose represses the expression of lac operon gene products

T

Label the highest, and lowest levels of the lac operon mRNA

A → No lac operon mRNA

C → Highest levels of lac operon mRNA

What is a Dioxic growth curve? What a flat part on it mean?

A growth curve, showing 2 different energy sources being used

Flat: Lag phase, where cell growth stops because the proper enzymes are being induced

T or F: the lac operon is fully off sometimes

FALSE

If it was, lactose wouldn’t be able to enter the cell and bind to the repressor protein (to promote transcription) when glucose supply runs out

Permease is needed to transport lactose, and is only in the membrane if the operon is on

This happens because the repressor protein is not bound 24/7, during periods it will dissociate and then reattach, and during these periods transcription can occur to make enough permease

What happens to mammals after weaning? But…?

Lactase enzyme production decreases when different diet components become introduced, which is influenced by environmental factors

But: A regulatory gene allows the lactase gene to be expressed all the time, relies on ancestors and how much lactose they consumed 

What is lactase

Enzyme that breaks lactose down into glucose and galactose

Why do the symptoms of lactose intolerance occur?

Excessive lactose (because it can’t be broken down) in intestines attracts water, which prevents water from being properly absorbed into the bloodstream and also intestinal bacteria ferments the lactose, and builds up

This leads to gas and diarrhea

What causes lactose intolerance?

Any mutation along the LCT gene

What is galactosemia? What is the key symptom? What are the damages?

Galactose in blood, individuals born without the enzymes needed to process galactose into glucose due to INHERITED mutations.

* Leads to a toxic accumulation of galactose, leading to organ and tissue damage
* Jaundice is key symptom that differs from lactose intolerance
* Brain damage from galactose build up, cataracts in eyes that block vision, kidney damage,

What are lactose free dairy products?

Just added lactase enzymes to break down the lactose

Who was Elie Metchnikoff?

First person to propose a microbiome, that good bacteria produces lactic acid, which is beneficial as it is bad for bad bacteria.

* Proteolytic bacteria is unable to grow at the low pH created by the fermentation of lactose

What are the 3 lactic producing bacteria? Why do they cause cheese and yogurt to form?

1. Lactobacillus
2. Lactococcus
3. Bifidobacteria

All ferment carbs in milk, and produce lactic acid as a product. The acid precipitates protein making solid material, and prevents the growth of bad bacteria

Why is yogurt and probiotics good for lactose intolerant individuals?

Microbial B-galactosidase is present in yogurt (it autodigests lactose), enhancing lactose metabolism in lactase deficient individuals

Probiotics colonize intestines, to increase b-galactosidase activity in feces, (but not in intestines) and help improve lactose digestion. Symptoms disappeared!!!!!

How does B-galactosidase survive the acidity of the stomach?

They have a protective cell membrane, and yogurt buffers the pH in the stomach

What is the development of a eukaryotic organism dependent on?

Communication between cells

* Signals exchanged between them
* Genes turned off/on