a functional structure composed of amino acids units joined together by pep tide bonds; large molecule composed of one or more amino acids chains. These amino acids are arranged in a specific order or sequence determined by the base sequence of nucleotides in the DNa coding.
Protein in models
When picturing proteins 3-D models are great because they indicate the topography; it 3D and has surfaces that can interact with others.It shows how it can mold itself or can fit together with other proteins.
a crevice in a protein that has a specific arrangement of atoms or amino acids that are able to bind to a substrate and undergo a chemical reaction; place where catalysis occurs.
Why do we have a three letter code for the amino acids?
We have a three letter code because the codons that are made from the DNA. Three nucleotide to code for one amino acids. The three letter amino acide code help to better line up the letters for the amino acid with the amino acid it creates.
- A sequence of three nucleotides that together form a unit of genetic code in DNA or RNA molecule; the nucleotides correspond to a specific amino acid or stop signal for protein
Different amino acid molecules react differently
all the reaction that take place in your cell involve a relatively small portion of amino acids ( mostly the polar amino acids)
Formation of a peptide bond cause the release of water and a little bit of energy; peptide bond is a dehydration reaction. peptide bonds help to form a polypeptide chain; is a dehydration synthesis
Properties of polypeptide bond
Rigid and planar; atoms (carboxyl and amine group) fall into the same plain. electron drawn down from the oxygen spend a lot of time around between the carbon and nitrogen via resonance; can form a partial double bond
- There is rotation (phi and psi) around the alpha carbon, but there is not rotation around
- Ramashandra plot diagram shows the limited degrees of rotation that the bonds can rotate. There is steric hindrance due to the size of the molecules that in the sidechains of the amino acids.The dots on the plot show where the protein sequences can fold. There is a double bond nature of a peptide bond and every third bond is rigid and planar. The actual shapes that a protein can form given certain amino acid sequences is limited.
each polypeptide not matter what the amino acid composition of it is, is going to have at least two charges at physiological pH
Linear order of the amino acids. Polypeptide back bone with side chain pointing in either direction; amino terminus to carboxy terminus.
- Polar side chains are outside and the hydrophobic( non-polar) side chains cluster on the inside, The driving force is hydrophobic hydrophillic interaction and this requires that no energy is used however the entropy in the world increased because the water molecules in the environment where very structure around the hydrophobic side chains in it unfolded form are no free to move around in the folded form.
- hydrophobic- hydrophobic properties help protein in folding.
- In a membrane the hydrophillic side chains cluster inside while and the hydrophobic sides chains are on the outside. ( hydrophobic tails, hydrophilic heads)
Noncovalent bonds(weak, reversible bonds)
- Ionic bonds, van der waals ( electron cloud interactions), and hydrogen bonds ( h and FON).
- Ionic interaction( can be two side chains are two atoms), are the strongest of the the interactions. Van der waals is the weakest and can occur between any atoms.
- Hydrophobic and hydrophillic interactions get the amino acids in the right place and fold. non-covalent bonds help to help hold them in the right( native) conformation.
- strong enough to hold things together but weak enough to pull things apart.
- Urea, heat, mercaptoethanol- are all agents which breaks the noncovelent bonds.
- Heat causes the atoms in the bond to move around rapidly making the bonds less stable.
- 50% of all proteins are able to refold on their own and others need heat shock proteins to assist them in refolding.
- denaturing- unfolding from native structure.
- Communicating within cell.The two main methods that proteins are able to communicate.
- How domain structure with a specific structure can be taken from one protein and plugged into another protein and the exact same function arises but a completely different end result happens( involving different pathways, generation of a different structure, couples with different protein)
- specific enzymatic structure that takes a phosphate from ATP and add it to a hydroxyl on a side chain; phosphorylating.
- In most cases this leads to the activation are the turning on of a protein as a result of the conformation change, but in other cases it can take a protein that is active and turn it off as well; and this is reversible.
Kinase domain example
CDK- cyclin dependent kinases- proteins that control the cell cycle they allow you to switch from one phase to the next within the cell cycle. A meeting point for input from a variety of different events or pathways within the cell. Once those particular events have occurred we want this centrall meeting point, the cyclin dependent kinase, to know these things have happened. This protein keeps track, and once all the events have happened this protein become active and gives the signal to move on to the next step.
steps to kinase interaction
- synthesis of cyclin
- addition of inhibitory phosphate
- the addition of an activating phosphate that gets the loop to move out of the way and expose the active site.
- a phosphatase to remove the inhibitory phosphate.
- Go from inactive form of CDK to active form
regulate mitosis. Controls whether or not the cell will divide. all the stages in the cell cycle controlled by these kinds of proteins.
- a protein that is also a tyrosine kinase.
- Has the same kinase domain that is in the Cyclin dependent kinase.
- There is 9 members in the Sarc family of proteins that are identified and coded in the human genome.
- Sarc protein couple or pair up with receptors. In the surface in the membrane of a cell is a receptor and to that recpetor a signal is going to come and a signal is going to bind and the information from that signal somehow wants to be transmitted to the inside of that cell to the nucleus to get some kind of change in gene expression. We want the signal coming from the outside of the membrane wants to affect the gene expression in the nucleus.
- These receptor often have kinase activity associated with them, however Sarc does not have that inherit kinase activity. SARC is an accessory protein anchored in the membrane ( anchorage indicated by the prenyl group attached to the end) . The SARC protein is inside the membrane so that when this receptor binds to its ligand the SARC protein will become activated. The SARC protein hears signals from other places leading to activation of the kinase domains. The Srac protein is able to detect the binding of the ligand by the the other domains that are connected tio the kinase domain( SH2 and SH3); SH2 and SH3 help to activate the kinase domain. SARC help to regulate things like cell adhesion development, and loss of SARC has been associated with various type of cancer.
Binds multiple prolines
Binding to phosphorylated tyropsine
Steps to get Sarc completely turned on.
- In it s inactive form, nothing is bound to the receptors.
- 1)Inhibitory phosphate bound to the SH2 domain; need phosphotase to remove the inhibitory phosphate that leads to release and conformational change
- 2) kinase needs to be activated to move the tyrosine in the active site of the kinase domain away. the tyrosine become phosphorylated and undergoes a confromational change that moves that protein out of the way.
- 3) A bind partner Nef has a domain that has multiple prolines in it
- receptor protein has a domain that has multiple prolinesit will be produced/ synthesized and will REPLACE the proline rich region within the sarc protein and binds to the SH3 domain that leads to a final conformational change.Same kinase domain that phosporylate a downstream target.
- receptor protein
- has a domain that has multiple prolines
- it will be produced/ synthesized and will REPLACE the proline rich region within the sarc protein and binds to the SH3 domain that leads to a final conformational change.
- The activity of the protein is dependent on whether or not it has GTP. It has a binding site for the entire GTP nucleotide.
- GTPases turn themselves off.
- Has its own internal activity for hydrolyzing GTP and GDP.
- Affect downstream events
Drosphila and the use of GTPases in the photorecptor of fruit fly eyes
- photorecpetors in fruitfly eye
- compound eye has 800 ommatidia
- each ommotidum is compose of 20 different cells : 8 photrecptor cells and 12 accessory cells that help photrecpot cells to work
- development follows a strict pattern.
- first cell to develop is R8
- R8- puts signals out in the form of transmembrane proteins; as cells develop around the R8 cell and they have receptor proteins in their membrane and the signaling protein binds to thee receptor protein on the adjacent cell and the adjacent cell turns into one of the other 7 cells. R7 develops last.
- Cell in fruit fly ommatidium
- Allows for the detection of UV light.
- without R7 ( sevenless or SEV)
- If you are missing the Sevenless gene( Sev gene) your R7 cells never developed.
- the way that the sev gene works is through an amplification pathway there are several of the proteins9 Rasss. SEVs) involved int he pathway that gets a cell to become a R7 cell.
what protein is called from the sevenless gene?
- The signaling molecule that the R8 puts out into its membrane is called BOSS( bride of sevenless).
- Protein coded for by the sevenless gene has a receptor tyrosine kinase (two functions: it is a receptor and it has a domain in the extracellular area that binds to a signal and once it binds to that signal it activates an activity where it will phosphorylate a tyrosine and that tyrosine is internal( it basically phosphorylates itself). the binds of BOSS to sevenless, then sevenless phosphorylating itself) starts of the signaling pathway tthat results in this cell turning from undifferentiated cell into a R7 cell. the signal has to get to the nucleus.
- Phosporylated tyrosines bind to SH2 domains.
- Down stream of receptor kinase is a protein that has SH2 and SH3 domains.
- SH3 binds to multiple prolines
SOS( son of sevenless)
- The binding of sos to sh3 domains leads to conformational changes that activates SOS.
- SOS is a GEF ( guanine exchange factor- a protein that stimulates the exchange of a GDP or GTP in a GTPase)
Guanine exchange factor- it is a protein that stimulate the exchange of GDP in a GTPase. and example of a GTPase is a SOS
- stimulated by the activation of SOS.
- A Gtpase to release its GDP and replace it with a GTP
- Rass is activated with GTP in its active site
- Rass is a diffusable protein that can diffuse around the cell.
- RASS will go on and it can effect other downstream events( i.e. the other kinases needed to get the signal to the nucleus and all the genes necessary to make a R7 cell gets turned on and all the proteins get made)
- RASS is activated by guanine exchange factor ( GEF ) Sos
We do not want RASS to be on all the time
- It will continuously influence downstream events and our bodies do not want this if it is not necessary
- RASS has to turn itself off and it has the ability to turn itself off. It can do this with with its GTpase activity however it needs help from GAP ( a GTPase activating protein) an accessory protein ..
- When GAp comes in contact with active RASS, it stimulates it GTPase activity, hydrolyzed GTP to GDP and turns off RAss
- We dont want Gap to turn Rass off initally, once the signal is already sent, we want the signal to survive for long enough to get the cell to start differentiating. SO therefore, Sev can temporary inhibit Gap but after a while
- GTPase domain is a part of RASS
- bind GTP and GDP
- when active affects downstream events
- GTP binding affects the switch helix ( loop ofd alpha helices) the position of the switch helix changes depending how many phosphates are in the active site of the GTPase domain.
- EF-TU is a protein that helps to make sure, in ribosomes, that the amino acid that is coming next in the correct one. it makes ure that there is not any mistakes in protein synthesis.
- This protein plus the other two domains that are attached to it sit onto of the amino acid that is attached to the tRNA and as the tRNA comes into the ribosome bringing the amino acid, this protein sits on top of the aminpo acid.
- If the amino acid is correct GTP is hytdrolyzes and the switch helix changes conformation and the interaction between it and the other domains change so that the protein opens up and it releases the amino acid and it can be added to the polypeptide chain.
Name two different pathways in which the same domain is used to produce a different outcome?
Compare and contrast GTPase signaling with phosphotase kinase signaling
Either there is a signal coming in from some source and that signal is heard by the kinase( in the chase of phophotase signaling) or the inactive GTPase.
- A collection of genes.
- entire DNA that codes to exhibit and perform functions that you need to do as a cell or organism.
- The percentage of your genome that codes for the protein that make us human is 1-1.5%.
- Genome is contained in the nucleus of the DNA.
What does the rest of your genome code for?
Beneficial characteristics of a genome
- Perform translations that are mistake free
- enable variability ( from person to person), but will not keep you from deleterious effects
- Deletion of a genetic mistake
- or completely remove( apoptosis)
- We want to be able to make mistakes because this allows evolution.
- you want the genome to be able to recognize itself.
Do we want out genome to be large or small?
- evolution has made the majority of the genome not code for anything even though the 98% does not code for anything and makes the genome a lot larger than it has to be.
- it harder to recognize a defective protein than a defective genome
What experiment was done to find out that the genome is contained in the DNA?
- Genome is in the nucleus of DNA and we know that through this exoeriement
- Strain of pathogenic bacteria causing pneumonia and it is characterized by growing it in a culture dish.They are called the S Strain, the smooth strain because thy are covered with a mucus like covering so you cannot see the topography; look smooth. they can give organism [pneumonia.
- while culturing the cellcan undergo random mutation to make a R (Rough)strain that cannot cause pneumonia; mucus coat was missing
- Scientist were able to turn them back R strain in the presence of heat killed S train and the daughters of the R strain were S strains and caused pneumonia.
- Scientist knew that it was DNA because the scientist broke down the heat killed S train into it marcomolecular component and then grew R strains with each of these molecules and realized that the only molecule that turn the R strain into the S train was DNA.
- Polymer of deoxoribonucleic units;
- linear structure consisting of a backbone connected to sugars that have the nucleotides base.
- double stranded
- No oxygen on DNA that makes it stable
- DNA is read from the 5` end to the 3`end.
- the sugar end to the phosphate end
- DNa is antiparallel
- AT and GC -> at the girls clubs
- helical form with bases facing middle
what are the differences between the base pairs ?
There are three hydrogen bonds holding together the GC base paris and two hydrogen bonds holding together the AT base pairs.
Characteristic to repair mistakes that are made in your genome ?
dependent on the amount of bonds between the AT and GC
bases sequences are hidden away in the middle of the structure
some places on DNA do not have alot of surface area for interactions
- There is a major groove and a minor groove.
- Most binding to DNA happens in the major groove because there are more places to attach.
- The packages form of DNA in chromosomes ; combination of DNa and equal mass od proteins.
- DNa has to be shrunk down in order to fit into nucleus and we do this by adding an equal mass of proteins
region of DNA at the beginning that initiates transcription of a particular gene
the use of a probe, which is the single stranded DNa that is complementary to the existing sequence of chromosomal DNA and has a fluorescent tag,
consists of exons , introns, and highly competatiove elements, and a 3` UTR ( untranslated region) and a 3`region that contains transcription factor proteins that bind to the promoter region and control rate of transcription
How do we shrink down DNA to fit into the nucleus ?
Add an equal wight of a protein
Genome break down
- introns( 25% and they have signals thus they are not completely useless and regulatory elements are hidden in introns thus genes can be controlled by them and there are more things to find)
- LINEs, SINEs, and repeatable sequences- (50% of genome , and are filled with viruss that got inserted and other invaders and through mutation has become useless)
- LINEs- long interspersed nuclear element. and SINE is short interspered nuclear element
- sigmental duplication and simple sequence repeat- ( ~10%)
- It is good to have a genome that is 98% useless because this way if an error occurs it will not affect the important parts. ( will not kill you )
- about 50 % of genome is called LINEs and SINES and they are basically invaders.Over the course of evolution different viruses and bacteria have invaded the genome and added DNA
evolutionary how can we use chromosome strucuture to see how related different species of organisms are ?
using conserved synteny and tracking the number of changes
- similar genes seem to be clumped together in little sections of DNA in chromosome and can be found in similar cluster in different chromosome in other organisms
- If the chromosome for this conserved syntenty is tracked by changes we can predict how far apart organism are evolutionarilty
NON PICTURE PERFECT DNA
DNA is usually diffuse and not compact like chromosome and this is because transcription factors need to be able to activate genes and the protein cannot get to them to transcribe them in the compact chromosome form
- Fold DNA on top of each other and help to compact DNA into the nucleus.
- How do nucleosomes do it?
- experiment: expose nucleosome DNA to nucleoase and realized that it only choped linker regions. discovered core particles that nucleosome core particle ( consist of core of protein withe DNA wrapped around it)
- want to get protein and DNA apart and used salt. this tells us that it takes a charge ( ionic bonds) to hold DNA to proteins. DNA has negative phosphate group on the backbone.
- The main amino acid protein responsible for the ionic bond are Lysine and Arginine( K and R) histones
- There are 8 proteins there that are actually 4 proteins twice. and these are at the core of the nucleosome.
- These protein are caled H2a,H3 aand H4 H2B ( these are the core particles)
- Xray crystalography shows nucleosome core particle with the DNA around it and we see that the proteins are in the core, but there are some that stick out. (IMPORTANT) and the wrapping has very little space, very compact.
- DNA from all organisms look like this. The histones of bean and cow look the same and they are very highly connserved evolutionarily.
- nucelosomes fold up into 30 nanometer units.
- Histones- highly alkaline protein found in euklaryotes that organize and package DNA into structural units called nucleosomes.
chops double stranded DNA
- allow proteins to come together and overlap
- the nucleosome is form is by step wise pairing. The N termin of the DNA stick out, why?
DNA wrapping around nucleosome
DNa wrapping around protein has to bend and found that the AT base pairs were the best to wrap around particles. This is because of the AT have less h-bonds and cannot strecth as much, this they favor the inner minor groves but GC can because of the more H-bonds and perfer the outer minor grove
After DNa wraps around protein, Histone H1 is able to wrap around the core particle and the DNA in the linker section and direct it. Histone H1 orient linking section to folding ontop of each other; hs 2 binding sites
What's up with the DNA taisl sticking out of the nuclesome core?
Tails bringing the compact strcucture together and helping to maintain eh compact structure of DNA. The tails interact,
- Formed when 8 seperaate histone units attach to the DNA molecule. The component tight loop of DNA and protein is the nucleosome.
- Multiple nucleosomes are coiled togther and these ends are stacked on top of each other.
- chromatin- fiber of packed nucleosomes , this is then looped and packages using other proteins
- 10,000 nuclei on tip of needle
- Chromsome ONLY FORM WHEN CELLS AR DIVIDING
What is the problem of packaging DNA in gene in nucleus ?
- all the proteins that interact with the DNA ( that fix them and so forth) do not have access
- thus the DNA must leave enough of itself packaged away as well as enough of itself exposed so that it ca interact with the necessary proteins.
Overall structure of DNA wrapped around protein. There are different types of chromatin based on how densely it is packed. as cells differentiate certain genes get packed away, exposed are half way between
Less densely packed and proteins have a chance to get to it
more heavily packed
Brownian motion of proteins is indicative of what?
Even though protein have the DNA wrapped around them in a nucleosome means that eh DNA is only going to be wrapped around them for a short period, then every now and then the weak interaction holding them together gives the DNA the potential to be released from the protein and then very rapidly re-wraps itself back around; always in motion. when the DNA is unwrapped in these sporadic times, proteins have the opportunity to interact with it ( non-histone proteins). they sneak in and lack on to sequences of genes that are important for transcription.
Chromatin remodeling proteins
take package DNA is that is wrapped around core histones and a portion of it grabs on to the linker region of the DNA and another portion that wedges in-between the proteins and the DNA of the nucleosome and using ATP hydrolysis to induce conformational changes and pry the nucleosome apart; dissociate a certain region of DNA from its packaging exposing a certain sequence sof genes. They can lead to removal of nucleosomes and completely free DNA and re-associate the nucleosome with DNA; help us to gain access to certain genes
what happens to the amino acid side chains that are on the tail of the core histone proteins?
- they can and do get post-translationally modified:acetylate, methylate . This leads to different characteristics of these proteins. One or two molecules per tail is on the histone tails, and it is the combinations that lead to different structure of DNA when they are read. This modification is done by enzymes and they can be seen by other proteins.
- The modifications also can be used as a signal for how densely the DNA should get wrapped . What type of chromatin.
comes in and has a specific binding site. This protein is designed to wrap around that tail and wrap very specifically to that post-translational modification of that tail.
large protein that Link together and interpret the reader proteins as they attach to their tails; this large protein holds together the reader proteins.
Code reader complex
- the interaction and entire structure 0of scaffolding protein and the reader proteins that It brings together.
- When reader proteins bind to the histone tails it results in a conformational change. That leads to the binding of other proteins., some of which could be chromatin remodeling complex or signals coming into the cell that expose genes through enzyme activation and remodel. Overal different code contained in the tails of histone proteins. Activate a variety of modfications.
- Attachment to other components in nucleus lead to gene expression, gene silencing, and othe biological fucntions
- Methylated K9=gene silencing
- (methylated and acetylated(K4, K9)) & ( acetylated lysine and phosphorylated serine(S10, K14)) = gene expression
- Methylated ( K 27) = silencing of the Hox genes, X chromosome inactivation.
mutations and mutation rates
how often is a mutation maintained!
carcinogens or mutagenes( like sunloight, smoking, chemicals) do not know what gene that they are attacking they directly attack DNA and cause changes in the amino acids,
The first step in gene expression where a particular DNA segment is copied into mRNA by the enzyme DNA polymerase.
Where ribosomes create proteins. The mRNA created in transcription is is decoded by the mRNA and used to produce specific sequence of amino acids chain.
Needs to be negatively charged so they can repel each other and not cause a clot.
How high is the rate of mutation
Very small because fast mutation will cause death.There are random mutation happening on our DNA an
From the beginning
Half DNA from Mother and the other half from you father. If you get a mutation in your somatic cell you get a disease if you get it in your germ-line cells, this is a mutation that you may give to your child.
Steps to replication ( complex like a engineer)
Pulse chase experiment (aka Faith experiment because we are labeling something we cannot see)
- Two phase technique use to understand cellular processes.
- The pulse phase is when the molecules are exposed to a labeled compound. This compound is incorporated into the process and
- Your are looking for something that you cannot see, specifically the formation of DNA.
- Label the molecules radioactively with a molecule called tritiumn. This helps us to see the DNA in a process, we label it radioactively because this is a very sensitive way to label DNA.
- We are going to incorporate that radioactive hydrogen into one of the building block of the DNA. tridium is an isotope of hydrogen. The thymidines, some of them, will be radioactively labeled. We get DNA replication started in a testtude with all the building block and the we will add( pulse ) radioactive thymidine. Tritiatred thymidine.
- Pulse last for a certain amount of time.
- Then we add the chase with regular thymidine to stop the process and run it on a GEL.
- Pulse no chase gives a radioactive band.
- pulse with chase will show two bands.
- This experiment showed that there are different sized DNA being produced.
- DNA grows in a 5`-3` direction.
What does the separating band on the gel of the pulse-chase experiment show you/display?
- The bands from the pulse stayed constant and and these represented the okazaki fragments of the lagging strand,while the bands that were climbing represented the leading strand.
- This shows the result of a moving process based on how long you pulsed and chased.
- This shows that the DNA replicated in slightly different fashions 9 lagging and leading strand)
Why do we replicated the DNA in two different fashions?
- Consider what we have and need to replicate:
- Free, 3` hydroxyl
- building blocks
- The template strand to tell what strand to make
- The enzyme that nakes DNA cannot start from scratch
Multisubunit enzyme that is used to unwind the double helix. This enzyme binds specifically to single stranded DNA. Helicase wraps around the single-stranded DNA using the energy of ATP hydrolysis uses conformational changes that propels it along the single strand until it get to double stranded DNA and begins, breaking the hydrogen bonds that keep the base pairs together. ATP hydrolysis helps to pry DNA double helix apart.
Once the particle are separated we want them to stay apart long enough for the new DNA to be synthesized !
Single-stranded region of DNA template with short regions of base pairs. Base pairs on the same single-strand of DNA are complementary and bind to each other. Internal base pairing; This looks like DNa helicase
- nucleotide from the single stranded DNA comes into the Dna polymerase and of that nucleotide is correct that triggers a conformational change in DNA Polym. that holds it in place. An this changee triggers the hydrolysis that triggers the removal(hydrolysis) of two of the phosphates from the the incoming deoxynucleoside triphosphate ( this drives the elongation of the newly formed strand).
- The two phosphates that get away are the pyrophosphate and they hydrolzed so they can be recycled into ATP.
- WE LOSE TWP HIGH ENERGY PHOSPHATE BONDS EVERYTIME A NUCLEOTIDE GETS ADDED TO DNA
- and once the pyrophosphate leaves this gets the DNA Polym to open up again. The conformational change of the DNA polymerase opening then bumps the backbone one nucleotide over so that the next nucleotide could be analyzed and encorporated.
syntheses new strand of DNA n the template strand will stop at double stranded DNA( programmed that way) when it encounter hair pin loop it will stop and that part of the DNA would never get replicated (not good)
What happens when DNA polymerase is stopped by hair-pin loop?
SSBP( single-strand binding proteins
- proteins that have domains that bind to the backbone of of single stranded DNA , these monomer of SSBP are polymerizing along the single stranded DNA. each monomer has two domains, domain A and domain B. Each domain has a binding site for the backbone of the single stranded DNA. They do not interact with the bases. It is sequence independent and can bind to anything regardless of sequences; can bind to anything and keep it single stranded.
- The core of the protein is a beta sheet with some alpha helices that
- HELP TO KEEP HAIR PIN LOOPS OUT OF DNA TEMPLATE
Why does DNA replicate in the 5'to 3` direction
Where do we start to replication , where does the 3`hydroxyl come from?
DNA polymerase( DR SAM VERSION)
- Is a DNA dependent DNA polymerase.The template that it uses and copies is DNA; It makes DNA;
- these enzymes cannot start from scratch, ,it needs the 3` OH to attach to.
- Polymerase does not make any mistakes
- DNA polymerase also stops at the RNA DNA hybrid that DNA primase primes.
- if the nucletotide that is wrong comes in the active site the conformational change is no make and the polymerase never closes and it is moved to a different active site( exonuclease active site) to be remove until the correct bp comes along.
- Is a DNA dependent RNA polymerase. It needs DNA as a template, but thge product that it is producing is RNA.The primer stretch is 10-12 ribonucleic acids not deoxy.
- They can start from nothing; These enzymes give us a stretch of 3` hydroxyl.
- Prinnase has room for error because they stretch of ribonucleic acids that it addes is going to be removed regardless.
- Adds another stretch of 3`OH primase every 200 nucleotides or so.
Gap in the phosphodiester backbone.
Short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication; short double stranded DNA sections.
joins together new Okazaki fragments in the growing chain; activity fixes (ligates) /links together fragments.
How does DNA ligasae does the linking?
- Uses ATP in 2 steps.
- One okazaki fragment has it free 2' hydroxyl and the other has the 5` end with the phosphate.
- Ligase latches onto knick, grab an ATP, and prime the 5` end of the okazaki fragment and along with the nucleotides adds a phosphate to the 5` end; the two phosphates are released as pyrophosphate.
- Then it bring the 3`Oh end in between the two phosphates and and link the 5` to the 3`. and AMP is released.
- DNA ligase is always looking to repair knicks
How tightly should polymerase hang on?
- Yes, because we want the bp to be sufficiently held and put together; no skipping, as continuous as possible.
- No, it would waste time at the knick;We want it to move on and synthesize more.as well as allow ligase to come in and solve the problem.
- This determines how long and tightly DNa polymerase holds on to the DNA when it is making DNA.Made up of a core of alpha helices that surround the DNA strands and beta sheets on the outside.
- This protein is responsible for holding polymerase on as it make DNA; It holds tightely as long as polymeriase is moving;
- Once as it stops moving it doesnt hold on any more.
- Requires the hydrolysis of ATP; doesnt clamp on by itself
CLAMP loader protein
- helps to get the clamp to the DNa polym.
- This protein is a pentameric that wedges into the grooves of the DNA and attracts the clamp protein.
- Clamp loader uses ATP to wrap around and once as it is wrapped around securely, it attracts DNA polym. ; the c;lamp holds on to the polymerase as it moves.
- The clamp loader lets go.
place where parent DNa is seprated into to strands.
Proteins involved in DNA replication
- They stay associated with one another. They helicase is associated with the primase.
- Helicase works 1000 nucleotide per second.
what happens if helicase makes a mistake? is it possible to correct? What kind of mistake?
Rare tautomeric form of C ( C*) happens to bp with A and is thereby incorporated by the DNA polymerase into the primer strand. This tautomeric C will then shift back to the normal C comforamtion and it can no longer base pair with the A. Now, there is a free three prime hydroxyl hanging because this false pair then dispair blocks further elongation of DNA polymerase. This need to be removed and DNA polymerase can chew backwards and find a proper base pairinf
- removing wrong nucleotide and make a 3`Oh available then polym cam continue it process.
- We are removing nucleotide 3` to 5` 9 backwards)
- the way that this happens happens with the conformationa change that happens to DNA polymerase.
Three ways to fix mistakes
prevent point mutations
- removes during replication process
- Base pair itself prevent mistakes
- exonucleoase proof reading
- cite-directed (mismatch)repair
why have research not found a polymerase that makes DNA in a 3 priome to 5` direction
- The nucleotides end up in the wrong direction;
- The active site of the incoming correct deoxynucleotide triphosphate has the phosphate energy on the opposite side.
- The energy that you would need to add this nucleotide to the DNA is on the wrong side and there is no way that it can be moved to the other side.
- This would encounter that every time that there is a mistake in the DNA.
- 3` to 5` would not allow proofreading thus it would not be able to fix mistakes and it would have to start over. Evolutionary, this was not advantageous and thus the processing from 3`-5` was throw out all together.
- This would mutate a lot more, the 5`-3` would keep up growth wise.
- an organism that does this couldn't keep up growth wise or mutated a lot more!
What happens if the wrong base-pairing is incorporated into the new daughter strand of the DNA other than stopping like what it did with the tautomeric C?
- First this looks like a little speed bump.
- This is fixed by two enzymes called Mut S and Mut L. Mut S find the mistake and the mismatched pair. Mut L determines what gets fixed,; these proteins are able to detect the mistake because tey look fo[r knicks, and knicks ar emore likely to be formed in the daughter protein.These proteins then remove the daughter strand that has the mistakes. They recongnuize which strand is wrong, new, and the daughter strand due to the kincks.
How do we accommodate/fix for knots when the DNA helicase is pulling part the DNa so quickly? How do we relieve the tension and prevent knots
- DNA topoisomerase!!!!!
- There are several types. They function ahead of replicatipon fork
- These have tyrpsine resideues in its active site that temporarily binds( covalently) to and break one of the backbones 9 a phosphodiester linkage)of the two strands. This frees up the other strands so that they can pin around that phosphodiester bond and this spinning redues the tension by spinning rapidly in the other direction; tyhe rotate relative to one another.
- Once the stress is relieve topoisomerase reattached the backbone. take same amount of energy to break as well as re-attach.
Where do we start replication?
- An origin of replication: to the machinery of DNA replication it recognized that spot. This start to replication forks. big chromosome more replication forks.
- For bothe prokaryotic and Eukaryotic organism
- 10,000 origins of repplication
Pulse-chase experiment that proved the origin of replication ?
- We stretch out DNA on microscope to make it linear
- Incorporate all machinery for replications( enzymes,etc)
- Pulsed with tritiaded tymidine( shows silver grains)
- First experiment no chase.
- ( take side with one daughter ds DNA expose to x-ray detector or film)
- Took a picture and saw that the Dna was repliation bi directionally away from a midpoint then in the second part of the experiment
- There was a chase and this showed a lighter labeling of the tritiaded thymidinea and showed that the DNA was replication away from a short center.
- If we want to prove what direction the Dna is replicating we have to do the pulse and chase.
- As DNa replication continued a lower rate of titirated thamindien appeared and it is shown on the X ray shot of the DNA is going.
- This proved that the replication bublke is bidirectional as well as which way the DNA is moving
series of proteins associated with identifying the origin of replication, they are refered to as Initiator protein. They recognize specific sequences in the prokarypotic genome and bind to those sequences. They then recruit helicase and the helicase is temporarily inhibited by a helicase initiator ( binding protein). Helicase inhibitor binds to these initators and once there is a strong attachment and there is a little bid og single stranded DNA created for helicase to bind unto, the inihibitor leaves and the lhelicase start to unwind the DNA