biochem MQ Flashcards
How many chromsomes do humans have
23 pairs of chromosomes
what are condensed regions of DNA called?
Heterochromatin
what did Treptococcus, Griffith and colleagues prove?
That bacteria can transfer genetic materail
What is the bacteriophage labelled with in the Hershey-Chase experiment
Sulfure-35
Does the number of chromosomes correlate with the complexity of an organism
No
Does the BCR-Abl fusion protein arise from chromosomal translocation
yes
does sickle-cell diease arise from a single nucleotide change in the alpha-globin gene
no
In the avery experiment did the DNAase treatment of heat killed smooth strain material result in transfromation of rough strain
No
Do mutations in the CFTR gene affect calcuim balance in the airway epitheluim
No
Why is the smooth strain of Streptococcus deadly to mice
Due to the ability to evade the host immune system
What purine base is contained within DNA
Adenine
What carbon of deoxyribose is the primary alcohol on
5
What properties of DNA did Rosalind Franklin and Maurice whilkins provide via their x-ray diffraction
double helix
phsphates on the oustide
What effect does high salt have on DNA melting
Increases melting temperature by neutralising negatively charged phosphate backbone
Is B-DNA is a right handed- helix
yes
What type of handed helix is Z-DNA
left
If the conservative model of replication would have been proven by Meselson and Stahl , what woudl you expected at the 1st generation
50% at the 14N density , 50% at the 15N density
What is the primary reason for the use of a DNA polymerase , such as Taq for PCR
Due to its ability to withstand high DNA denaturing temperatures
What is required for next generation sequencing?
Polymerase chain reaction
Nucleotides with a 3’ reversible blocking group
what subunit stabilises the assembled E.Coli RNA polymerase
omega
What subunit directs the E.Coli RNA polymerase to the correct trasncription initaion sites?
sigma
Where are the signals for termination
in the newly synthesised RNA
What does the poly A tail do
Increases stability and assits in nuclear export
What does the 5’cap added to MRNAS in eukaryotes contain?
7-methyl-cytidine
What is the splicesome comprised of
protein and snRNA
What do miRNAS do and whats their aim
miRNAS destabilise mRNAS eitjer leading to their degradation or blocking translation , the aim is to block protein production
What did Avery prove about DNA
Proved that DNA caused the transformation
How many hydrogen bonds between pairs AandT and GandC
A-T=2
G-C=3
What are the histones in chromatin?
H1,H2A,H2B,H3,H4
What is the role of H1 histone
its a linker histone and helps compact DNA
What tells the polymerase when to stop?
Termination signals
what enhances and blocks promotors?
enhance-activators
block-supressors
What is an operon
A group of genes that are trasncribed together to produce a single messenger RNA molecule that encodes multiple proteins
What are genes called that are expressed all of the time
constitutive expression
what are genes called that are expressed under certain conditions
regulated expression
What does allolactose do?
Binds to the repressor and prevents it from binding to the operator so that the gene cant be transcribed
allolactose is an inducer of the lac operon , binds to the lac repressor protein, which is normally bound to the operator region of the lac operon, preventing transcription of operon genes
what is an operator
an operator is a DNA sequence that controls the transcription of genes by binding to regulatory proteins
What does microRNA do
Bind to specific mRNA and leads to degradation of mRNA
What are the characteristics of fibrous proteins?
insoluble , silk-web, intracellular cytoskeleton, extracullar matrix
what are the charactertics of globular proteins?
soluble, exact 3d shape is critical in their fucntion
how many different side chains are used in proteins?
20
what are the 20 different amino acids?
glycine
alanine
cysteine
phenylalanine
methionine
tryptophan
valine
leucine
isoleucine
proline
serine
threonine
asparagine
glutamine
tyrosine
aspartic acid
glutamic acid
lysine
arginine
histidine
what are the non polar amino acids
glycine
alanine
valine
cysteine
proline
leucine
isoleucine
methionine
tryptophan
phenylalanine
what are the amino acids with a positive charge
lysine
arginine
histidine
what are polar amino acids?
serine
threonine
tyrosine
asparagine
glutamine
What are the amino acids with a negative charge
aspartic acid
glutamic acid
why do non-polar groups like to cluster together?
Because of their inability to form H-bonds , they group together because of a need to avoid water
What did Brenner and Crick suggest?
suggested that each codon must contain three bases
Where do release factors bind to
They bind to the A site
what three stages is translation divided into
initiation
elongation
termination
What did Linus Pauling and Robert Corey try to do?
They tried to work out the structure of the fibrous protein a-keratin using X-ray diffraction data
What three rules did they(Linus and Pauling)have to follow to fit the data?
1-there can be no rotation around the planar peptide bonds
2-other parts of the chain must be felxible
3-structure must have the maximum number of stabilising forces between residues and stabilising forces must be indepdent of primary structure
why is the anti parallel beta sheet more stable than parallel
Anti -parallel is more stable as it contains more H-bonds and the H-bond atoms are aligned directly opposite making for stronger bonds
what is calmodulin
calmodulin is a protein that binds to calcium and regulates processes
how many calcuim binding sites does calmodulin have and how many different proteins can it bind to?
4 calcuim binding sites
can bind to two different proteins
How does calmodulin activate a protein
by binding to them and inducing a conformational change that allows the protein to interact with other signaling molecules
What are the two important uses of DNA polymerase reactions
-DNA amplification(PCR)
-DNA sequencing
How do you amplify DNA (PCR)
-Denature to produce SS DNA
-Add short primer (complementary)
-Lower temp to anneal, add DNA polymerase to join back together
-Add thermostable DNA polymerase , required for DNA extension
what are ddNTPs
moelcules used in DNA sequencing that terminate action of DNA polymerase
What is the gel Agarose used for
To seperate double stranded DNA fragments
Would DNA run to the positive or negative electrode during electrophersis
Positive end (Due to DNA being negatively charged)
what are the steps of next generation sequencing
-Oligonucleotide adaptor annealed to short fragment of DNA
-Use of PCR to create copies
-Use of flourescent nucleotides for sequencing
-Use of reversible terminator
What are rNTPS
monomeric building blocks that are used to synthesize RNA oligonucleotide chains
What are promotors
DNA sequence that initiates the transcription of a gene
What do promtors in prokaryotes contain
-A conserved AT-rich region at around -10 (Pribnow box)
-A consensus sequence (TTGACA) at -35
What do termination signals do
Tell the polymerase when to stop
How many different RNA polymerases do euakroytes have
3
what does RNA polymerase 1 do in eukroytes
ranscribe the three largest ribosomal RNAs (rRNAs) that make up the ribosome’s basic structure(18s , 5.8s and 28s)
How is pre-MRNA processed
-Addition of a polyA tail to the 3 end of the RNA transcript ( increases stability)
-Addition of a cap to the 5 end
What are genes called that are expressed all of the time
Constitutive expression
How do you estimate the molecular mass of a protein
Multiply number of residues by 100
What direction is the mRNA read in
The 5 to 3 direction
What is the movement from triplet to triplet called
Translocation
What mutations occur when there are errors in the DNA/mRNA sequence
wild type
Mis-sense
Nonsense
Silent
Frameshift base deletion
What did Linus Pauling and Robert Corey do
tried to work out structure of the fibrous protein a-keratin using X-ray diffraction data
-Their work on a-keratin led to the discovery of the alpha helix
–They proposed that a-keratin is made up of two aligned alpha helices twisted into a coiled coil
How does calcuim activate a protein
-Binds to calmodulin
-Causes its shape to change
-Binds to a protein activating it
What does the Andinsen experiment on Ribonuclease show
The folded, active form of protein has the lowest free energy
All of the information needed by a protein to fold to this structure is encoded in the primary structure
Not all proteins fold as easily
Some require protein disulphide isomerases (PDIs)
What breaks electrostatic bonds
heat,pH and salt
What is the pKa of the ionsing group of an amino acid side chain
is the pH where 50% ionisation has occurred
What factors affect the pKa
polarity of environment , absence or presence of water and nearby charges
What makes/breaks salt bridges and ionic bonds
changes in pH
-Causes the charge on side chain to appear/disappear
what are protein folding funnels
Series of increasingly restricted movements until a low energy minimum is reached
Protein folding funnels are a theoretical model that shows how a polypeptide chain folds into a protein
What are the factors that disrupt protein structure
-Heat
-pH
-Ionic strength
-Denaturing agents( organic solvents, chaotropic agents)
-Uv
What bonds are weak in the tertiary structure
hydrogen bonds and hydrophobic interactions
What bonds are strong in the tertiary structure
electrostatic and covalent
what proteins catalyse phosphoroylation
kinases
what are the two main categories of kinases
Ser/Thr kinases and Tyr kinase
what removes a phosphate group from a protein
phosphatases
How does chromatin become transcriptionally active in terms of acteylated chromatin
Due to removing the acetyl groups from the lysines of histones , which loosens up the chromatin and the gene is now turned on
Is chromatin compact in deacetylated chromatin
yes its compact and transcriptionally repressed so gene is turned off
Where does glycosylation occur
In the golgi and ER
What is glycosylation
The addition of complex carbohydrate group
What is palmitoylation
The addition of hydrophobic groups (fatty acids) to proteins
What is ubiquitination
The post -translation modification process that attached a ubiquitin protein to a target protein
Are the processes of : modifications of amino acids and proteolytic cleavage irrerversiable modifications or reversible
irrerverisble
what is proteolytic maturation
Proteolytic maturation is a process that involves the cleavage of proteins at specific sites to create mature proteins and functional enzymes
what are zymogens
They are precursors of enzymes that are activated in a proteolytic cascade after release as granules into the duodenum
Zymogens (also known as proenzymes) are inactive enzyme precursors that require a biochemical change, such as cleavage of one or more peptide bonds, to become an active enzyme.
What are precursors
a precursor is a substance that is inactive but can be converted into an active enzyme
What is a homomeric network
a network of proteins made up of multiple identical polypeptide chains, or repeating subunits ( all the same subunit)
What are heteromeric networks
a complex made up of multiple different proteins
Would the affinity be higher of lower with more hydrophobic interactions
Higher affinity
what do interactions in proteins consist of
charges or hydrophobic groups
What do oligomeric intercations of proteins require
Complementary interfaces
how do microtubules form
when protein dimers, made of alpha and beta tubulin, join together to create long, tubular polymers
What does Beta tubulin hydrolyse into
GDP
What is PKR (protein kinase)
It is a kinase that is activated when double stranded RNA is present in the cell
What is the role of Myelin
has a lot of lipid present so acts as a good insulator
Structure of phospolipids
-Glycerol backbone
-Phosphate attached to glycerol and an alcohol attached to the phosphate
-2 Fatty acids attached to glycerol
Structure of Sphingolipids
-Different acetyl chain to phospholipids
-Has an amine group instead of glycerol
-Major component of neural membrane
How do lipids form in the membrane
The aceyl chains cluster together because they dont like to interact with water, formation of lipid bilayer due to presence of water
What occurs in the crystalline phase for lipids
Cis bonds in the lipids prevent the close packing of acyl chains resulting in a bilayer where the acyl chains are mobile
-In the liquid crystalline phase the lipids move around in the plane of the bilayer at biological temperatures
What is flip flopping of lipids
the process of moving lipids from one side of a cell membrane to the other
What is laterla diffusion
The ability for lipids to move around rapidly , spinning round
What are the two types of proteins
Integral membrane proteins
Peripheral membrane proteins
What are the major membrane proteins
-Channels (ion)
-Transporters(amino acids)
-Receptors
-Structural proteins - important in maintaining tissue integrity
What do trans-membrane helices do in order to produce a path for movement
They cluster together to produce a poalr route across the bilayer
What occurs in a gated ion channel to allow and stop movement through
-In the closed state the large leucine residue point into the centre of the channel blocking any acess of sodium
-In the open state , leucine slides away from the middle of the centre , this is do the acetylcholine binding and initaing a conformational change so leucine begins to slide away
Are channels hydrophilic or hydropobic pores
Hydrophilic pores
What are the problems involved with studying the membrane
-The integral membrane proteins are held together by lipids so in order to study them , we have to disrupt the membrane to extract the membranes , disrupt lipid bilayer
What does the detergent do , used to study the membrane
It forms a micelle around the hydrophobic parts of the protein, hydrophobic interactions between detergent and hydrophobic region of protein
–lose 50% of protein due to this detergent
How areHydropthay plots used to predict the structure of a membrane protein
by analyzing its amino acid sequence and the hydrophobicity of its amino acids
Is Phosphorylation post translational modification
Yes
What do the membrane proteins B-Barrels form
hydrophilic pores in the outer membranes of bacteria and mitochondria
How can pores be produced in membranes
By Beta Barrel structures
What are PTMs
Post translational modifications
What do PTMs do
are chemical modifications of proteins that can trigger an action at specific steps in the cell cycle
What do PTMs include (Post translational modifications)
-Proteolytic cleavage of regulatory subunits or degradation of entire proteins
-Covalent addition:phosphorylation/methylation/glycosylation
-Think of it like attaching a tag , chemical fixed modification
What PTMs (post translational modifications do proteins need to do in order to become functional
-Fold
-Associate to other proteins (as dimers fro example)
-Undergo enzyme-catalysed chemical modifications of the protein structure (known as post translational modifications)-labels attached to it
How does insulin become its active form
-Insulin synthesised as a pro insulin
-Disulfide bridges form between the two red strands (the bottom strands)
-Active form of insulin due to the cleavage (cutting)
What is Ubiquitination
a post-translational process that modifies proteins with ubiquitin
What does phosphorylation of a protein do
Alters the charge , structure and dynamids of it
Does acetyl-lysin have a neutral or positive charge (page69 picture)
Neutral charge
What does the post translation modification lipidation do
Regulates membrane trafficking,protein secretion,signal transduction,apoptosis
membrane targetting,regulation of protein function,regulation of protein-protein interactions
what are polypeptides linked by
linear polymers linked by peptide bonds
What is starch made up of
made up of amylose(1,4 linkage)and amylopectin(1,4 and 1,6 linkages)
What is hemicellulose and what is its structure
-Different types of biopolymers present in the cell wall
Forms rigid microfibrils
Less rigid than cellulose
-Beta 1,4
what are enantiomers
each of a pair of molecules that are mirror images of each other
what are epimers
carbohydrates which vary in one position of for the placement of the OH group
-Differ in the configuration of only one chiral centre
What are anomers
Type of epimer that differs in their 3D orientation at the anomeric carbon
How does glycosylation occur
Glycosylation (addition of glycans)is a process catalysed by enzymes known as glycoside hydrolases (GHs)and glycosyltransferases(GTs)
What are Gts - Glycosyltransfereases
-GTs are enzymes that catalyse the formation of the glycosidic linkage from activated glycoside donors
-Use a sugar donar
-Binds to growing sugar
What do GHs do -Glycoside hydrolases
GHs are enzymes that catalyse the hydrolysis of the glycosidic linkages (enzymes that catalyse the hydrolysis of glycosidic bonds between sugar molecules in glycosides) .Retaining GHs cleave the glycosidic bond and maintain the stereochemistry of the anomeric carbon , hydrolysis mechanism involves the formation of a covalent enzyme substrate intermediate at the anomeric carbon, while inverting GHs lead to a product with a different stereochemistry ,The product formed by an inverting GH has the opposite stereochemistry at the anomeric center compared to the substrate.
What is N-glycan synthesis
complex carbohydrate structures, known as N-linked glycans, are attached to proteins
What do glycoside hydrolases do
Remove monosaccharides
What do glycosyltransferases do
Add monosaccharides
what are the three types of protein glycosylation
-N-glycans
-O-glycand
-Glycosaminoglycans(GAGs)
What are the 6 classes for enzymes
1-oxidoreductases
2-transferases
3-hydrolases
4-lyases
5-isomerases
6-ligases
What does the active site of an enzyme contain
Binding and catalytic residues
What are the two features of substarte specifcity
-Shape filter
-Binding affinity
What are co-enzymes
organic molecules which provide/remove groups for reactions called co-substrates
They assist enzymes in catalyzing biochemical reactions by binding to the enzyme and helping to facilitate the conversion of substrates into products. Coenzymes often act as carriers of specific atoms or groups of atoms during these reactions.
How do you work out the specifc activity of an enzyme (whats the formula)
enzyme activity/total amount of protein
What does V represent in terms of enzymes
Reaction velocity
What does a small Kd value mean in terms of enzymes
a high affinity
what does Kd represent in term of enzymes
affinity for substrate
What is Vmax
It is when all of the enzyme are enzyme substrate complexes
what are isozymes
Different enzymes , same substrate
what three factors do you compare when comparing enzymes
1-Turnover number
2-Efficiency
3-Potency
what is enzyme potency
How many times faster a reaction is with the enzyme
What does kinase do to an enzyme
-kinase attaches a phosphate group which distorts the shape of active site , active site turned off as no longer complementary
-phosphatase reverses this , so active site is turned on
Does a v type enzyme affect binding or catatylsis
catalysis
Does a k type enzyme affect binding or catalysis
Binding
what is meant by cooperativity with enzymes
Its where the substarte binding to one site increases the affinity at another
What do allosteric enzymes do
Its where the substrate binds to an allosteric site (not on active site) and changes its shape , increasing its affinity
how do suicide substartes (irreversible inhibitors )work
(molecules look similiar enough so it can bind but doesnt produce the right product , enzyme cant bind to normal substrate due to change in active site) This is permanent and enzyme is now damaged so wont work
what do uncompetitive inhibitors bind to
The enzyme substrate (after substrate has binded)
What is the transition state for an enzyme
is is the halfway stage between enzyme substrate and enzyme product
What are the ways to incraese enzyme reactions
a)position the reactant correctly for interaction(pointing in same direction , massively raising conc of substrates in two active sites)
b)distort the reactants making them less stable (making them more likely to react)
c)stabilise transition state (must promote formation of transition state)
d)change the environment to favour the reaction (for example change pH)
what are the examples of serine proteases
-Trypsin
-Chymotrypsin
-Elactase
-Thrombin
They all do the same reaction , break peptide bond
-They have different substrates (bind to different amino acids within protein), theres binding specificity
What is the mechanism action(the 6 step process) for serine proteases
The mechanism of action for serine proteases follows a well-known, multi-step process that involves the serine residue in the enzyme’s active site. Serine proteases, such as trypsin, chymotrypsin, and elastase, catalyze the hydrolysis of peptide bonds in proteins by using a catalytic triad consisting of serine, histidine, and aspartate
1-nucleophilic attack
2-formation of tetrahedral group
3-cleavage and loss of c-terminal fragment
4-Nucleophilic attack on polypeptide carbonyl by water
5-Covalent intermediate
6-Cleavage and loss of N-terminal fragment
what are the basic steps of gene cloning
1-gene isolated
2-inserted into vector
3-transported into host cell
4-multiplication of recombinant DNA
5-Division of host cell clones
what vector is most commonly used for gene cloning
plasmid vector
For gene cloning , how is the gene isolated
using PCR
What does PCR require
-Double stranded DNA template
-Pair of oligonucleotide primers
-Dna primers
-Nucleotides
What are the steps of PCR
1-Denaturing template DNA to single stranded DNA with heat
2-Lower temperature to allow primers to anneal
3-DNA polymerase extends the primer
What are basic steps to insert recomimbant DNA into a vector
1-Plasmid vectors
2-Restriction enzymes to cut DNA
3-Gel electrophoresis to separate DNA fragments
4-DNA ligase to join DNA fragments
What do restriction enzymes do
Restriction enzymes recognise specific sequences of bases in the DNA , binds to these sites and cuts the double stranded DNA by hydrolysing the phosphodiester bond
What are the two types of restriction enzymes
1-Exonucleases-Removing nucleotides from the ends
2-Endonucleases-Break nucleic acid chains in the interior
Will smaller fragments of DNA move further down agarose gel
yes
What enzyme is used to join DNA fragment to plasmid vector
DNA ligase
How does DNA ligase work
-Sticky ends are complementary so form hydrogen bonds
-Reaction catalysed by DNA ligase which catalyses a phosphodiester bond, between the sticky ends and plasmid
-Most experiments use T4 DNA ligase which requires ATP
How does blunt end cloning occur
-Cut plasmid and PCR product using different enzymes
-They dont come together due to not having comp base pairing
-This mean we have to use another enzyme to cut off the sticky end to create blunt ends which are now complementary
How does using DNA polymerase to fill in the end of the vector occur
-3 different restriction sites
-Instead of cutting off the overhangs we can instead fill them in using DNA polymerase and dNTPs to create blunt ends
How does using linkers and adaptors to insert recombinant DNA occur
-PCr product has short piece of DNA stuck onto it - blunt end joining
-Pink part has got a restriction site in it which is compatible with plasmid
-Can then stick restriction site onto each end of DNA
-Need to do blunt end joining to get the adaptors onto DNA
-Have to cut off phosphate group on adaptors so this means it lacks a phosphate group so no phosphodiester bond
-Use polynucleotide kinase to add a phosphate group and can then carry out sticky ended ligation
What does TA cloning consist of (using terminal transferase)
-Put nucleotides on the ends of the PCR product (adenine onto each end)
-Terminal transferase adds thymine onto the vector , onto the blunt ends
-created sticky overhangs
How do you prepare competent E.Coli cells
-Treat normal bacteria with cacl2 which modifies it by changing the charge , makes it more likely to bind to negative DNA
-Heat shock it which makes the membrane more fluid , puts energy into the membrane , making phospholipids more mobile , so DNA is more likely to be taken up the cell
-Then grow transformed cells onto agar plate
How do we distinguish bacteria that have taken up the recombinant DNA or self ligated vector? - the three examples
-Replica plating
-Blue/white selection
-Restriction mapping
How does replica plating occur (in order to distinguish bacteria that have taken up recombinant DNA)
-Tetracycline is a selectable marker
-The selectable marker helps us distinguish between recombinant and self ligated vectors
-Cuts the middle of the tert resistance gene
-Any plasmid that successfully then incorporated gene of interest is no longer going to have functioning tertr gene
-All those that have self ligated will have amp + tet
-Have a second plate that contains tetr, and stamp the ampliicin plate and transfer in onto tetr plate and stamp it
-In the replica plate , any bacteria that has our gene of interest wont have tetr resistance , they will die
How does blue/white selection occur
-Dont have tetr resistant gene
-Have the Lac Z gene- encodes the code enzyme beta galactosidase and this converts lactose into galactose and glucose
-Those with self ligated bacteria will have a functioning Lac Z gene so will produce the Beta galactosidase enzyme
-Streak these colonies out onto ampicillin plate
-Look for a colour change - with functioning Lac Z gene
How does restriction mapping occur
-Transform ligation mix into bacteria
-Plate out on agar and ampicillin
-Grow up bacteria and extract DNA
-Using restriction enzymes to distinguish between recombinant and self ligated vector
What is needed to produce protein from a cloned gene
An expression vector
What are expression vectors
Sequences that are required for transcription
Why is bacteria used to be the host cell over animal cell
-It divides quickly
What is an advantage of using animal cells as the host cell rather than bacteria
-Animal cells glycosylate proteins whereas bacteria doesnt
how do you determine the overall charge of the polypeptide sequence
To determine the overall charge of the polypeptide sequence, we need to count the basic (positively charged) and acidic (negatively charged) amino acids. Each basic amino acid will contribute a +1 charge, and each acidic amino acid will contribute a -1 charge.
draw the structure of all 20 aa
what is the three letter and one letter code for glutamine
Gln,Q
