Principles of replication, transcription, translation and their regulation Flashcards
Life can look many different ways, what is the main structure that is the foundation for life? Why?
The plasma membrane. It consists of lipids and proteins that provide a sturdy yet pliable, hydrophobic barrier to the outside world. The plasma membrane helps with cellular integrity throughout the whole life cycle and blocks charged/polar molecules from entering. It is selective in what it takes in.
Cells also have the cytosol on the inside, an aqueous solution of soluble molecules (like RNA, enzymes, cofactors) and ions. Most cells also have a compartment for their genome; nucleoid/nucleus.
Explain the difference between aerobic and anaerobic conditions.
aerobic - with oxygen.
anaerobic - without oxygen.
Organisms living in aerobic conditions obtain energy from transferring electrons from fuel molecules to oxygen, while anaerobic organisms transfer electrons to other electron acceptors, like nitrate (forming N2), sulfate (forming H2S), or CO2 (forming CH4).
Organisms can be classified by their energy source. Explain the difference between phototrophs and chemotrophs.
Phototrophs trap and use sunlight to obtain energy and carbon, and chemotrophs derive their energy from oxidation of a chemical fuel.
Describe the biggest differences between animal and bacterial cells.
Bacteria have no mitochondria and no nucleus, a circular genome and have different membrane structure than animal cells, like containing a peptidoglycan. Animal cells are generally much bigger and have extensive compartmentalization and membrane enclosed organelles, with a less rigid phospholipid bilayer plasma membrane.
Plant cells are very similar to animal cells, but additionally have a cell wall and chloroplasts.
Biochemistry is both a biological but also a chemical science. What does all four of the “molecules of life” have in common?
That they are polymers of smaller monomers:
- Proteins are polymers of amino acids
- Nucleic acids are polymers of nucleotides (nitrogenous bases + sugars)
- Carbohydrates are polymers of sugars
( -Lipids are polymers of hydrocarbons)
The structural hierarchy: the cell and its organells > supramolecular complexes (held together by non-covalent interactions) > macromolecules (held together by covalent bonds) > monomeric units.
What is important to keep in mind when studying biomolecules in vitro?
In vitro studies have of course revealed a lot of important biochemical information, but isolated components can (and will) behave a lot different in vitro than when it can interact with the rest of the cell components in vivo.
What four elements most abundant in living cells? Why?
The four most abundant elements in living organisms, in terms of percentage of total number of atoms, are hydrogen, oxygen, nitrogen, and carbon, which together make up more than 99% of the mass of most cells.
They are the lightest elements capable of efficiently forming one, two, three, and four bonds, respectively; in general, the lightest elements form the strongest bonds.
Name five important functional groups occurring in biomolecules, and how they are structured.
- Carbonyls: aldehyde R-C(=O)-H, carbon with a double bound oxygen or ketone R-C(=O)-R.
- Amino group R-N+H3
- Disulfide R-S-S-R
- Thioester R-C(=O)-S-R
- Phosphoanhydride R-O-P(-O neg, =O)-O-P(-O neg, =O)-R. (breaking this bond generate a lot of energy).
- Mixed anhydrides (very reactive) R-C(=O)-O-P(-O neg, =O)-OH
All biochemistry follows the first law of thermodynamics: energy can not be created nor destroyed. Explain how energy from sunlight can be used to maintain DNA integrity.
The energy from sunlight is used to reduce carbon dioxide, and the energy released from this reaction is stored in covalent bonds in glucose. Grucose can then be oxidized in glycolysis (and in the cirtic acid cycle) and the potential energy in the bonds is used to phosphorylate ATP, or transfer electrons to electron carriers which through the ETC drives ATP synthase. The energy stored in the bonds of ATP can then be coupled to energetically unfavorable reactions to drive them, such as maintaining DNA integrity.
The energy could be converted to several types of work, like chemical transformations, heat, metabolism (producing compounds simpler than original –> higher entropy), all of which result in decreased entropy by polymerization, or information storage.
How does cells perform reactions with a positive delta G (endergonic reactions)?
By coupling them to exergonic reactions (with a larger negative delta G), so that the overall reaction becomes exergonic which provides enough energy that can be used to carry out the endergonic reaction.
Example, the phosphoanhydride bonds in ATP breaking is an exergonic reaction, that is often coupled to endergonic reactions such as DNA synthesis to supply enough free energy to make the endergonic reaction favorable.
Explain in short how enzymes work.
Enzymes are biocatalysts that lover the free-energy required to get over the activation barrier for specific reactions, without being consumed in the process.
They do this by providing a more “comfortable” fit for the transition state, a surface that complements the transition state in stereochemistry, polarity, and charge. The binding of enzyme to the transition state is exergonic, and the energy released by this binding reduces the activation energy for the reaction and greatly increases the reaction rate.
A further contribution to catalysis occurs when two or more reactants bind to the enzyme’s surface close to each other and with stereospecific orientations that favor their reaction. This increases the probability of productive collisions between reactants and leads to the reaction being a looot faster than the uncatalyzed reaction.
Life evolved in water. What makes water a good solvent?
Water molecules are polar and with its almost tetrahedral structure (the two hydrogens and the two free electron pairs) it has the ability to form four hydrogen bonds. This is what makes liquid water to be stable over a large temperature span and make it a powerful solvent as it can stabilize ions, and polar biomolecules.
Even though non-covalent interactions are weaker than covalent ones, The non-covalent binding of a substrate to an enzyme can be very stable, why?
Enzymes and their substrates interact by more than one non-covalent interaction, for example, it could interact by several hydrogen bonds and one ionic interaction with additional interactions through hydrophobic effect and van der waals interactions. Even though these are all individually weak and only last for a very short period of time, all of these would need to dissociate at the same time for the substrate to get unbound, which is highly unlikely. So the cumulative stability of non-covalent interaction is much higher than their individual strength summarized!
The energy released when an enzyme binds noncovalently to its substrate (through many weak interactions) is the main source of the enzyme’s catalytic power.
Explain in short what acids and bases are (Bronsted-lowry).
Acids = proton donors and bases = proton acceptors.
What does the pKa value say about an acid?
Low pKa = strong acid, high tendency to donate its proton(s).
High pKa = weak acid, low tendency to donate proton.
What does the “p” in pH stand for?
“p”=the negative logarithm of. So pH means the negative logarithm of [H+] (in M), at neutral pH, the concentration of H+ = 10^-7 = pH 7 because log (1/10^-7)=7.
How does buffer systems work?
Buffer systems consists of a conjugate acid-base pair that can “neutralize” small changes in pH (small additions of weak base or acid). They do this by reacting with the proton released from the acid or with the base. A good example is HAc (CH3COOH), which can ionize to form H+ and Ac- (acetate: CH3COO-), with it’s own equilibrium constant. When H+ is added the protons can be taken up by Ac- to reform HAc, and when OH- is added, an H+ can be donated from HAc to form acetate and H2O. This results in very minor changes in overall pH.
Conjugate acid base pairs have a buffering capacity +- 1 around their pKa, so HAc for example have a pKa of ~4.7, which means it has buffering capacity between pH 3.7 and 5.7.
Give one example of a buffering system of cells.
One example of a buffering system is the side chain of histidine residues of proteins, which are weak acids with a pKa of 6. In natural pH, they are deprotonated and below 6 they are protonated, so they are an effective buffering system at natural pH.
If a weak acid is added and the pH falls, the side chain can take up the H+ and neutralize the pH. Fine tuning of pH is very important, as enzymes in different compartments have different optimal pH.
Some other examples: organic acids buffer the vacuoles of plant cells, ammonia buffers urine, bicarbonate buffering in the blood which regulate the rate of respiration (s. 326) and the phosphate buffer system, which acts in the cytoplasm of all cells, consists of H2PO−4 as proton
donor and HPO2−4 as proton acceptor:
H2PO(^−)4 ⇌ H+ + HPO(^2−)4
Since DNA is complementary, one strand is in 5’->3’ direction while the other is in 3’->5’ direction. What consequence does this have for replication?
Since the strands are anti-parallell and replication only occurs in a 5’->3’ direction, one strand is replicated continuously (leading strand) and one is replicated discontinuously (lagging strand) in the opposite direction as the replication fork. The fragments are called Okazaki fragments (that are then ligated together by DNA ligase)
DNA replication is semi-conservative, what does this mean?
That the two daughter DNA molecules contain one parent stand and one daughter stand, each parent strand is the template for the daughter strand.
Can DNA replication start anywhere in the genome?
No! Replication always start at an Ori (origin of replication), the number of Ori in a genome differs between species.
What are the two types of nucleases called and what do they do?
Endo- and exonucleases. Endonucleases cleave and degrade nucleic acids (DNAses if specific to DNA) in the middle of the DNA molecule, while exonucleases degrade DNA from either of the ends of the DNA molecule, either in a 3’->5’ direction or 5’->3’ direction.
Restriction endonucleases selectively cleave at specific sequences (some only where modification has happened, like methylation), very important in molecular biology and biochemistry.
What enzyme catalyzes DNA replication? Explain the reaction in short.
DNA polymerase.
The reaction carried out by DNA polymerase is a phosphoryl (PO4^-) transfer, where the OH group of the 3’ end of the growing strand performs a nucleophilic attack on the alpha-phosphorous in the deoxynucleoside-triphosphate (A, G, C or T), forming a covalent bond between the 3’O and the nucleoside, elongating the strand and releasing an inorganic pyrophosphate (PPi). All DNA polymerases requre 2 mg2+ ions as cofactors. The deprotonation of OH -> O- is mediated by one of the mg^2+ the other mg2+ binds to the incoming dNTP and facilitates the departure of PPi, which in turn are stabilized by asp residues.
Each nucleotide added cost the equivalent of 1 ATP (GTP, CTP, TTP), so this is a very energy demanding process!
Besides cofactors and substrate, DNA polymerases need something else to function, what?
They need a free 3’-OH group to start the catalysis, so a primer is needed. The primer is usually an complementary RNA molecule that is synthesized by specialized enzymes (eg. primase, a type of RNA polymerase) that synthesize primers when and where they are required.
Why are deoxynucleotides (dNTPs) used in DNA synthesis instead of NTPs?
dNTPs are used in DNA replication instead of NTPs because dNTPs have a deoxyribose sugar, lacking a 2’ hydroxyl group, which provides stability to the DNA structure. DNA polymerases specifically recognize and incorporate dNTPs, ensuring high fidelity in replication. Incorporating NTPs, which have a 2’ hydroxyl group, would disrupt the DNA double-helix structure and decrease replication accuracy.
What is meant by “processivity” of an polymerization enzyme?
Processivity describes how many monomers (nucleotides in the case of DNA Pol) on average are incorporated in the growing chain before the enzyme dissociates from/falls off the template, and lets you compare the rate of polymerizing enzymes.
Different DNA polymerases vary greatly in processivity.
Explain in short how DNA polymerases are structured.
Most DNA polymerases are shaped kind of like a human hand gripping the DNA, with a thumb and fingers. The palm of the “hand” contains the active site which in turn consists of two parts, the insertion site; where a new dNTP is added, and a postinsertion site. When the phosphate bond brakes, the enzyme moves forward, so that the inserted dNTP is moved into the postinsertion site.
DNA polymerases have extremely high fidelity (accuracy). In E. Coli a mistake is only made about every 10^9-10^10 nucleotide, and considering that the whole genome is in the 10^6 order, this means it has extreme fidelity! What three things makes this possible?
- The optimal hydrogen bonding of A=T and G≡C only accounts for a part of this high fidelity.
- Geometry in the active site also contributes. The shape of the active site is constructed so that only the correct pairings fit well, so if for example A is in the template, a G or C doesn’t fit quite right sterically and therefore is not incorporated. An incorrect nucleotide may be able to hydrogen-bond with a base in the template, but it generally will not fit into the active site.
- The biggest contributor to the high fidelity is that DNA polymerase also have “proof reading” in the form of 3’-5’ exonuclease activity in a position close to the active site. So after each base is added, the enzyme repositions so that the base is in the exonuclease site, which is highly specific to incorrect matches. If the pairing is incorrect, the enzyme wont be able to position back and the phosphodiester bond is hydrolyze the the mispaired nucleotide and then repositions back to add the correct one.
- Besides all this, there are also many DNA repair enzymes that go through the DNA to find and correct mistakes.
Note: Some DNA pol doesn’t have proof reading activity, eg Taq polymerase. Wonder how Thermus aquaticus survives without high fidelity?? Question for another day.
Compare DNA pol I, II and III in terms of processivity and polymerization rate.
DNA pol III, responsible for replication, has a processivity of over 500 000 and a polymerisation rate of about 250-1000 nts per second! It has two beta-clamps that grip both strands of DNA, with lagging strand looped, which increases the processivity by several orders.
The others are mainly for DNA repair and have much lower polymerization rate (10-40) and fidelity (3-1500). DNA pol I in E.coli also have 5’-3’ exonuclease activity, which enables it to produce a nick where it finds an error, break down part of the strand and synthesize a new one to repair the error in a process called “nick translation” (also inportant to remove primers)
DNA polymerase alone is not enough to efficiently and accurately replicate DNA, the replisome of E. Coli consists of DNA pol III + 20 other proteins/cofactors. Name three of these and explain what they do.
- Helicase: rips open the dsDNA to make the template strand accessible.
- Topoisomerase: Relieves the strain on the surrounding double helix caused by the unwinding around the replication fork.
- Primase: Synthesizes the RNA primer needed for the polymerization to be started.
- DNA ligase: Covalently closes/seals nicks between the Okazaki fragments in the discontinuous strand.
- DNA-binding proteins stabilize the separated strands.
