Manufacture and secretion of proteins

This is how proteins are manufactured and secreted in cells.

Amino acids pass through the plasma membrane by diffusion down a concentration gradient or by active transport. They then combine with their specific tRNA. They are then transported to ribosomes attached to the rough endoplasmic reticulum, where they are incorporated into a polypeptide via protein synthesis. The protein molecule is then packaged into a transport vesicle, which pinches off from the rough endoplasmic reticulum. The vesicle transports the protein molecule to the Golgi apparatus where it fuses wiht the cis face of the Golgi apparatus. Protein is chemically modified as it moves through the Golgi apparatus, undergoing post-translational modification. Trans face gives rise to vesicles containing the inactive form of the protein. The secretory vesicle eventually buds off from the secreting face of the Golgi apparatus. When vesicles reach the cell surface, it fuses with the plasma membrane, and releases the inactive protein by exocytosis, using ATP in the process.

Respiration

Respiration is the breakdown of large glucose molecules into simple molecules such as carbon dioxide and water with the simultaneous release of energy inside living cells. It is an enzyme mediated process.

Main processes:
Glycolysis (glucose to pyruvate, occurs in cytosol)
Link Reactions (pyruvate to acetyl CoA, occurs in intermembranal space)
Krebs cycle (acetyl CoA to carbon dioxide, occurs in the matrix)
Oxidative phosphorylation (occurs in the inner membrane)

Calvin Cycle

The Calvin cycle is also known as the light independent reactions, as they can occur in the dark. It occurs inside the stroma of the chloroplasts. There are three stages: carbon dioxide fixation, reduction of carbon dioxide, and regeneration of ribulose biphosphate.

Carbon dioxide is initially accepted by ribulose biphosphate to become a 6 carbon unstable intermediate, which breaks down to form two molecules of 3 phosphoglycerate. This reaction is catalysed by RuBP carboxylase, or Rubisco. 3-phosphoglycerate is later phosphorylated by a molecule of ATP to form 1,3-biphosphoglycerate and ADP. 1,3-biphosphoglycerate is later reduced by NADPH to form phosphoglyceraldehyde (GALP) and NADP. 1/6 of all GALP formed is channelled to form sugars and other useful substances for the plant. The remaining 5/6 of GALP undergoes a series of chemical reactions and is phosphorylated by ATP again to regenerate RuBP.

Q: Describe semi-conservative replication

A segment of DNA is unwound by helicase. H-bonds between both strands are broken. Primers are laid down by primase in the 5' to 3' direction. DNA polymerase III adds complementary free nucleotides to 3' growing end.

Link Reactions

The Link Reaction is a process of respiration and occurs in the intermembranal space of mitochondria. It involves the oxidative decarboxylation of pyruvate to form acetyl CoA. NAD is reduced to form NADH and a proton.

Genetic terms

Genotype is the genetic composition of an organism.
Phenotype is the set of characteristic manifested by an organism.
An individual has a heterozygous condition if he has 2 different alleles of the same gene
An individual has a homozygous condition if he has 2 identical alleles of the same gene.
Codominance is a genetic interaction where the codominant alleles of a gene express themselves equally in the phenotype
Dominance is a genetic interaction where 1 allele of a gene masks the expression of another allele in the heterozygote.
Locus is the position of a gene in a DNA molecule of a chromosome
A gene is the functional unit of inheritance
An allele is an alternative form of a gene.

Stem cells

Stems cells are unspecialized cells that are capable of continually renewing and dividing through cell division for long periods. They are also capable of differentiating into specialized cell types under appropriate conditions. Therefore the two normal functions are to reproduce and give rise to more cells indefinitely (in the case of 3 to 5 day old blastocyst), and to regenerate multiple types of cells to replace those that have died or were damaged (where there is a local infection/injury).

Polymerase Chain Reaction (PCR)

There are three stages to PCR: denaturation, heating, and extension.

In the first stage, denaturation, the reaction tube in the thermal cycler is heated to 95 degrees Celsius. The high heat denatures DNA by breaking the hydrogen bonds; the double helix is broken into single strands. In the second stage, annealing, the temperature is cooled to 60 degrees Celsius to allow DNA primers to form H-bonds with their complementary bases at 3' end at both strands. In the third stage, extension, the temperature is increased to 72 degrees celsius. Taq DNA polymerase binds to DNA template where primer has annealed. Complementary nucleotides are added to 3' end of both primers, thus catalysing synthesis of a new daughter strand. Chain reaction occurs as the products of previous reactions are used as the reactants in the next cycle.

Q: List the advantages and limitations of PCR

Advantages:
PCR is highly sensitive as it is capable of amplifying sequences from minute sequences of target DNA. It also has high fidelity as it produces the sequences required with a low error rate. It is also rapid as it can generate many copies within a short period of time. It is a quick and reliable method for detecting all mutations. Moreover, amplification can be done from unusual sources e.g. Egyptian mummies where the DNA quality is not good due to degradation.

Limitations:
Some knowledge of DNA/amino acid sequence of desired gene/protein is needed for the synthesis of flanking primers.
Non-target DNA sequence may be amplified instead, as primers are short and there is the possibility of many sequences complementary to primers. Also, Taq polymerase does not perform proofreading so there is the possibility of errors

Human Genome Project

Benefits:
It provides an extremely detailed map of the entire human genome which elucidates the genetic basis of diseases, hence allowing for the early detection of genetic diseases and improved diagnosis. The project enables novel molecular treatment such as gene therapy to be devised to cure genetic diseases.
Ethical Concerns:
It may detect genetic diseases early thus causing those affected to feel like social outcasts.
Employers and insurance agents may abuse the information, hiring workers based on genetic predisposition.

Q: Explain the functions of primers in PCR

Primers mark out a sequence of DNA to amplify. This sequence would be complementary to the target DNA strand which it attaches itself to. They also prevent the single stranded DNA from re-annealling by sticking to a part of the target DNA. They also allow Taq polymerase to work from a 5' to 3' direction, since the enzyme can only work with a free 3' end.

Q: Explain the effects of too high a temperature on an enzyme

If the temperature is too high, an increase in kinetic energy of the molecules will cause excessive vibrations of the molecules. This will disrupt the 3 dimensional structure of the enzyme, causing it to become a random coil. The enzyme is therefore denatured. The active site of the enzyme will be "lost" as it is no longer complementary to the substrate in size, shape and charge, thus the enzyme becomes nonfunctional as the enzyme-substrate complex cannot be formed.

Glycolysis

Glycolysis the breakdown of a glucose molecule into two pyruvate molecules. It is an example of substrate level phosphorylation. It occurs in the cytoplasm, whether in aerobic or anaerobic conditions.

Glucose > Glucose-6-phosphate > Fructose-6-phosphate > Fructose 1,6 biphosphate > 2 x phosphoglyceraldehyde > 2 x 1,3-biphosphoglycerate > 2 x 3-phosphoglycerate > 2 x phosphoenolpyruvate > 2 x pyruvate

NB
Glucose > Glucose-6-phosphate: 1 x ATP is used
Fructose-6-phosphate > Fructose 1,6 biphosphate: 1 x ATP is used
These two steps are known as the energy investment phase.

1,3-biphosphoglycerate > 3-phosphoglycerate: payoff of 1 x ATP per molecule (total 2 ATP)
2 x phosphoenolpyruvate > 2 x pyruvate: payoff of 1 x ATP (total 2 ATP)
These two steps constitute part of the energy payoff phase.

Net gain: 2 ATP per molecule of glucose
Also note that NAD is reduced to form NADH when phosphoglyceraldehyde reacts to form 1,3-biphosphoglycerate.

Q: Explain how protein synthesis can be controlled at a transcriptional level in prokaryotes

In prokaryotes, genes that encode enzymes of a metabolic pathway are usually clustered together on the chromosome in a region called an operon. A typical operon consists of regulatory sequences, like promoter, operator, terminator, and structural genes. Lac operon is an inducible operon that can be under negative control by lac repressor or positive control by CAP. In the absence of lactose, repressor binds to operator and blocks RNA polymerase from binding to promoter, thus no / low level of transcription of structural genes. In the presence of lactose, lactose will bind to repressor and change its conformation and prevent repressor from binding to operator, thus RNA polymerase can bind to promoter and transcription occurs. When both glucose and lactose are present, bacteria can selectively catabolise glucose instead of lactose. In the absence of glucose, the concentration of cAMP is high and cAMP binds to CAP and activates CAP, thus CAP binds to CAP-binding site and enhances rate of transcription. In the presence of glucose, concentration of cAMP is low and CAP not activated, thus CAP does not bind to CAP-binding site and transcription of structural genes occur as a low / basal rate.

Enzyme Inhibition

In competitive inhibition, inhibitor has a shape, charge, size and structure similar to that of the substrate. It therefore competes with the substrate for the active sites to form the enzyme-inhibitor complex (E-I complex) and reduces the number of active sites available for the substrate to bind and form the enzyme-substrate (E-S) complex. If substrate concentration is less than that of competitive inhibitor, it is more likely for the enzyme to collide with the competitive inhibitor and form E-I complex, so rate of product formation will decrease.
Km will increase and there are less number of active sites available for substrate molecules. If substrate concentration is more than that of competitive inhibitor, it is more likely for enzyme to collide with substrate and form E-S complex. The reaction will eventually reach Vmax but only at high substrate concentration. One example of competitive inhibition is where malonate competes with succinate for succinate dehydrogenase.

For non-competitive inhibition, the inhibitors have a structure different from that of the substrate. It binds away from the active site at the allosteric site, inducing conformational change of active site. It prevents substrate from binding to the acgtive site as it is no longer complementary to the shape, orientation, and charge fo the substrate, hence the E-S complex cannot be formed. Km remains constant, affinity does not change. Vmax is lower and there are fewer functional enzymes. for example, cyanide is a non-competitive inhibitor of oxygen to cytochrome oxidase.

Q: Compare and contrast the structure of collagen and cellulose

The monomer of collagen is an amino acid whereas the monomer of cellulose is beta-glucose. The monomers in collagen are linked by peptide bonds whereas those of cellulose are beta (1,4)-glycosidic bonds. Collagen is an alpha helix polypeptide with a turn every three residues whereas cellulose is a straight chain with adjacent glucose molecules rotated 180 degrees with respect to each other. Collagen has hydrogen bonds between its chains (glycine on one chain and hydroxyproline on another strand), these hydrogen bonds account for its strength. Likewise, cellulose is a strong material because of the hydrogen bonds between the -OH and O of adjacent molecules. Collagen's macrofibre structure is that of triple helices lying parallel with covalent bonds at staggered ends of carboxyl and amino terminal, thus forming collagen fibre; in cellulose, chains of beta-glucose associate in groups to form micelles, micelles arrange in larger bundles to form microfibrils. Microfibrils combine by cross-linking of chains to form macrofibrils. Both collagen and cellulose are resistant to chemical change because of their stable macro structure

Chromosomal Aberration

Chromosomal aberration refers to a change in number of chromosomes, can occur on one, or several, or all of the chromosomes within a nucleus. This is cause by the failure of sister chromatids to separate during anaphase of mitosis or the failure of homologous chromosomes to separate during anaphase I and/or II of mitosis. Aneuploidy refers to the loss or gain of single chromosomes but polyploidy refers to the increase in entire haploid sets of chromosomes. Structural aberrations are the result of chromosomal breaks that occur during meiosis or mitosis. This often leads to reshuffling of alleles on the chromosome. This is often caused by the deletion, duplication, translocation, inversion of several gene loci within chromosomes.

Q: Explain how mitosis ensures that cells are genetically identical

During S phase of interphase, semi-conservative replication of DNA occurs. Complementary base pairing rules are adhered to (on complementary strands, A is paired with T; C is paired with G). During anaphase, sister chromatids are separated and move to opposite poles of the cell. New cells have the same number and kind of chromosomes as their parents.

Mitosis

Prophase
Chromatin condenses to form chromosomes. Centrioles migrate to opposite poles of the cells (except in plant cells which do not have centrioles). Microtubules develop and form a star shaped structure known as an aster, and spindle fibres may develop. Nucleolus disappears, and the nuclear membrane disintegrates.

Metaphase
During this phase, the chromosomes arrange themselves at the equator of the cell, and they are attached to the spindle fibres at the centromere. Each chromosome is still composed of two chromatids.

Anaphase
During this phase, each centromere duplicates and the spindle fibres contract or shorten further. This causes the chromatids of each chromosome to separate and migrate to opposite poles, led by the centromeres. The shortening of the spindle fibres is due to the progressive removal of the tubulin molecules of which they are made.

Telophase
Chromatids reach their respective poles and a new nuclear envelope forms around each group. Chromatids uncoil and lengthen. The spindle fibres disintegrate and a nucleolus reforms again in each nucleus. A new cell membrane is formed. In the case of plants, a new cell wall also forms.

Cytokinesis subsequently occurs.

Membrane systems

Endoplasmic reticulum (ER) consists of a series of membranes creating channels within the cytoplasm, which are continuous with the nuclear membrane. They also form sheets that enclose cellular spaces called cisternae. ER provides a large surface area for chemical reactions. Rough ER is studded with tiny granules (ribosome) that may be seen with an electron microscope. As a result, rough ER is most abundant in cells that either secrete proteins or are growing rapidly (eg cells in the pancreas that secrete insulin). Unlike rough ER, smooth ER has no ribosomes. Its function is to synthesize and store steroids as well as lipids. Hence it is abundant in cells that secrete steroid and lipid substances (eg cells in the sebaceous glands of the skin.) ER also forms a structural skeleton for maintaining cellular shape.
The Golgi apparatus consists of a series of parallel membranes that enclose flattened fluid filled spaces called cisternae. The ends of the membranes form vesicles that pinch off as Golgi vesicles. The Golgi apparatus is responsible for the manufacture of glycoproteins, the secretion of carbohydrates, the transport, modification and storage of materials such as lipids, the formation of lysosomes, and the post-translational modification of proteins such as enzymes in cells. (Think of it as the "warehouse" of the cell)
The cell surface membrane has numerous functions. It is a selectively permeable membrane that has a phospholipid bilayer that has proteins and cholesterol embedded within the bilayer, as well as extrinsic (peripheral proteins). The lipids and proteins on the membrane also can rotate about their own axis, hence it is fluid in nature. As a result, the cell membrane has a "fluid-mosaic model". It serves as a regulatory barrier to control the transport of substances in as well as out of the cell. The hydrophobic and hydrophilic portions control the entry and exit of fat soluble and water soluble molecules respectively, so it helps ot maintain ion concentrations. It provides shape and structural support to the cell. Cholesterol also prevents the phospholipids from packing tightly together, making the cell membrane more flexible and stable. The cell membrane also maintains the ionic balance in the cell with reference to its surroundings via active transport processes such as sodium potassium pumps (removing sodium ions while accumulating potassium ions via active transport). Water soluble ions are also transported by facilitated diffusion by channels across the membrane. Glycoproteins function as antigens to allow cells to be recognized by other agents, e.g. enzymes, hormones, and antibodies. Carbohydrate chains (glycolipids) attached to the surface of the membrane act as recognition sites.

Structure and functions of organelles

The nucleus is bound by two membranes known as the nuclear envelope, which has perforations called nuclear pores. Chromatin within the nucleus condenses to form chromosomes during cell division. The nucleus controls all cellular activity. During mitosis, it also undergoes division so that cell replication may occur. It contains the DNA and produces RNA. Within the nucleus is the nucleolus, a body rich with ribosomal RNA. Its function is to produce ribosomes
Mitochondrion is an elongated structure with two membranes. The outer membrane is separated from the inner membrane by the intermembranal space. The inner membrane is also highly folded to form extensions known as cristae, which increase the surface area for respiratory processes to take place. On the cristae are stalked particles, which protrude into the matrix. The stalked particles allow the flow of protons back to the matrix of the mitochondrion, acting as the driving force to combine ADP with inorganic phosphate to form ATP.
The lysosome is a small spherical structure containing digestive enzymes. These enzymes enable it to undergo autophagy (digest cytoplasmic organelles and other membranes), exocytosis (release its enzymes out of the cell), and autolysis (self-destruction of the entire cell itself). They are only found in animal cells.
Ribosomes are composed of two subunits, and they assist in protein synthesis by forming polyribosomes along the mRNA.
Chloroplasts contain the enzymes necessary for the Calvin cycle in the stroma. They also contain chlorophyll and other pigments that are required for the light reactions on the thylakoid membrane (PS 1 and PS2). They also store photosynthetic products in the stroma(glyceraldehyde-3-phosphate is subsequently converted to starch grains).
Centrioles are the apparent organizers of the mitotic spindle, and they are only found in animal cells. They consist of a pair of cylindrical structure that are found in teh centrosome. Each centriole is composed of 9 groups of microtubules. Microtubules radiating from the centrioles form the spindle fibers during mitosis.

Q: Explain how a foreign DNA fragment can be introduced into a plasmid that produces blunt ends.

Cut the DNA fragment and plasmid using the same restriction enzyme. Add guanines to DNA fragment blunt ends and terminal transferase, to produce a single chain of guanines to both the blunt ends of the plasmid vector. Add cytosines to vector blunt ends, to produce a single chain of cytosines to both blunt ends of the DNA fragment.
Mix the cut plasmid and cut DNA fragment together for recombination to occur. Single chain guanine will bind to single chain cytosine by complementary base pairing. Add ATP and DNA ligase to seal the nicks by forming phosphodiester bonds in the sugar backbone.
Add recombinant DNA to bacteria culture for transformation to take place. Use heat shock and add calcium chloride to facilitate uptake of recombinant DNA.

From here on, answer is question specific...
Culture bacteria in agar plate containing ampicillin (assuming original plasmid had ampicillin resistant gene). Bacteria that has taken up the plasmid vector, whether reannealed or with the foreign DNA will be resistant to ampicillin and grow on the agar plate. ...

Q: compare and contrast the prokaryotic and eukaryotic genomes

The eukaryotic genome is diploid but the prokaryotic genome is haploid. The eukaryotic genome has multiple origins of replication but the prokaryotic genome has only one origin of replication. The eukaryotic genome may have more than one chromosome but the prokaryotic genome has only one chromosome. The chromosomes in eukaryotic cells are linear but the chromosome in prokaryotic cells is circular. The eukaryotic genome size is much larger (10 to 100000 million bases) but the prokaryotic genome size is much smaller (0.6 to 10 Mb). The eukaryotic gene length is around one an a half times as long as the prokaryotic gene length. The eukaryotic genome is much more complex, with distinct regions such as the centromere and the telomere, whereas the prokaryotic genome is much simpler with no distinct regions. In the eukaryotic genome, the DNA is bound to histone proteins to form nucleosomes, which wind around each other to form chromosomes, but in the prokaryotic genome, DNA is bound to nucleoid associated proteins to form a DNA-protein complex. The eukaryotic genome is enclosed within a true membrane bound nucleus but the prokaryotic genome does not have a true nucleus, only a region called the nucleoid.

Q: Outline the differences between prokaryotic control of gene expression with the eukaryotic model

In the prokaryotic model, the regulation of transcription is by the control of operons. However, in the eukaryotic model, it is via the control of transcription factors. In prokaryotes, transcription can occur simultaneously with translation as there is no compartmentalisation by a nucleus. In eukaryotes, after transcription, RNA processing has to occur before translation of the mRNA. Transcription is carried out by 1 type of RNA polymerase in prokaryotes, but it is carried out by 3 different RNA polymerase enzymes in eukaryotes. Enhancers are absent in prokaryotes as promote regions on the DNA are regulated by operators, but in the eukaryotic model, enhancers are present to regulate the initiation of transcription from a promoter.

Control Elements

Control elements are non-coding DNA regions that regular transcription by RNA polymerase II. They come in two forms: enhancers and silencers.
Enhancers are a short region of DNA that can bind to proteins called activators. When such binding happens, transcription of a gene will be initiated. The gene can be some distance away from the enhancer, or even on a different chromosome. The increase in transcription is due to activators recruiting transcription factors, which enhances the binding of RNA polymerase.
A silencer is a DNA sequence capable of binding transcription regulation factors termed repressors. Upon binding, RNA polymerase is prevented from initiating transcription thus decreasing or fully suppressing RNA synthesis.

Q: Explain why mutations for antibiotic resistance spread so rapidly among bacteria

Frequent use of antibiotic kills many bacteria, creating a selection pressure. As a result, bacteria with mutations that give them resistance to a particular antibiotic have an advantage over the other bacteria that do not. They survive to reproduce more than the other types, and pass on this advantageous allele in greater numbers. The frequency of this allele therefore increases in subsequent generations, leading to an increase in frequency of resistant types in subsequent generations. As bacteria have a haploid genome, this alleles will be expressed and there will be no masking of any recessive alleles. In addition, bacteria multiply rapidly as they reproduce asexually by forming clones.

Gene mutation

A gene mutation is a change in one or several bases. A base may be added, deleted, or substituted with another base. This is caused often through the action of damaging chemicals, radiations, or through errors inherent in DNA replication and repair reactions. If a base is added or deleted, frameshift is the result as a different amino acid is coded for. As the sequence of amino acids is altered, a non-functional protein may be produced. However, in some cases, mutations do not affect the amino acid coded for. This is because the mutation may occur in a non-coding region of the DNA. The genetic code is also degenerate as more than one codon may code for a certain amino acid. Finally, the amino acid may be in a non-essential position of the polypeptide, hence its 3 dimensional structure is not significantly altered.

Q: Discuss the role of membranes in the synthesis of ATP during photosynthesis

The chloroplast is enclosed by two membranes, and this separates reactions within the chloroplast from the rest of the cytoplasm. The double membrane of the chloroplast also serves to maintain high substrate concentrations within the chloroplast for photosynthesis. The outer membrane of the chloroplast is smooth and is freely permeable to many substances. The inner membrane regulates the entry ot substances needed for photosynthesis, such as water. Floating in the stroma are tiny membrane sacs known as thylakoids. The thylakoid membranes are the site of the photosynthetic light reactions. Photosystems 1 and 2, are sequentially organized on the thylakoid membrane, responsible to trapping light energy for photosynthesis. Electron carriers are also organized on the thylakoid membrane. The flow of electrons down the electron transport chain provide the energy needed for the active transport of H+ ions from the stroma, across the thylakoid membrane, into the thylakoid space. The hydrophobic nature of the thylakoid membrane functions as a channel for the diffusion of H+ ions. The diffusion of H+ out of the thykaloid space provides the energy for the synthesis of ATP from ADP and inorganic phosphate. ATP synthase in the stalked particles catalyses the reaction.

Q: Explain how the structure and properties of triglycerides and phospholipids are related to their functions in living organisms.

Triglycerides are a good source of energy as they have a high ratio of energy storing carbon-hydrogen bonds to carbon atoms. They are also an important source of metabolic water as the triglyceride molecules yield metabolic water on oxidation. This is particularly important to desert animals such as camels. As they are poor conductors of heat, they also function as thermal insulators by preventing excessive heat loss. This is especially important for mammals living in cold climates, such as polar bears. Fourth, triglycerides are a good storage molecule as they possess low mass thus much can be stored in a small volume. This makes them especially useful for animals where locomotion requires mass to be kept to a minimum. As triglycerides are large and uncharged, they are insoluble in water, thus they can be stored in large amounts without having any effect on the water potential of cells. Finally, triglycerides are less dense than waters, hence providing aquatic mammals buoyancy.
Each phospholipid molecule consists of a hydrophilic phosphate head and two hydrophobic fatty acid tails. In aqueous environment, phospholipid molecules form a bilayer in biological membranes. The phosphate heads face outwards and interact with the aqueous medium, while the hydrocarbon tails face inwards and are shielded from the aqueous medium. This allows the formation of a hydrophobic barrier between the interior and exterior of the cell. In addition, carbon-carbon double bonds at fatty acid chains causes a kink and results in greater separation between molecules and membrane fluidity. Hydrophobic interactions occur between the lipid layer and hydrophobic portions of membrane proteins, holding membrane proteins of various functions in place. Hydrophilic heads interact with hydrophilic portions of peripheral proteins, loosely to the holding them at the surface of the membrane. Phospholipids associate covalently with membrane carbohydrates to form glycolipids, which function in cell-cell adhesion and recognition.

Q: Explain the small yield of ATP from anaerobic respiration

In the absence of oxygen, the conversion of pyruvate to acetyl CoA, the Krebs cycle, and oxidative phosphorylation are prevented as oxygen is not available as the final electron acceptor.
Hence, only glycolysis can occur, generating 2 molecules of ATP by substrate level phosphorylation by breaking down glucose into pyruvate. Glycolysis can occur even in the absence of oxygen as the oxidising agent of glycolysis is NAD, not oxygen. A sufficient supply of NAD is available as the NAD reduced during glycolysis is regenerated by transferring electrons from NADH to pyruvate during the fermentation process.

Q: Describe the role of NAD in aerobic respiration

NAD is a coenzyme and an electron carrier, it acts as an electron acceptor in glycolysis and Krebs cycle through dehydrogenation processes and is then reduced to NADH. WHen it is reduced to NADH, it is utilized as an electron donor during oxidative phosphorylation. Electrons are transferred from NADH and FADH2 to oxygen by the electron transport chain via a series of redox reactions. The electrons are gradually passed down to lower and lower energy levels. The energy is used to generate a proton gradient across the inner mitochondrial membrane. This stores potential energy that can be used to phosphorylate ADP to ATP during chemiosmosis.

Q: Outline the main features of a Krebs cycle

The Krebs cycle occurs in the matrix of the mitochondrion. It occurs only in aerobic conditions, that is, only when oxygen is present. Its main function is to oxidise acetyl CoA generated from glycolysis. In the Krebs cycle, acetyl CoA and oxaloacetate undergo a condensation reaction to form a six-carbon compound. Through a series of decarboxylation and dehydrogenation reactions, intermediate 5-carbon and 4-carbon compounds are formed with the release of carbon dioxide. 1 ATP molecule is generated per turn of the Krebs cycle by substrate level phosphorylation; hence 2 ATP molecules are formed per molecule of glucose. Most fo the chemical energy is transferred during the redox reactions where the electron carriers NAD and FAD are reduced to form coenzymes NADH and FADH2, respectively. The Krebs cycle is also cyclic as the starting product, oxaloacetate, is regenerated.

Q: Distinguish between competitive and non-competitive inhibition of enzymes

A competitive inhibitor is structurally similar to the substrate but a non-competitive inhibitor is often structurally different from the substrate. Hence, a competitive inhibitor will compete with the substrate for an active site but a non competitive inhibitor will bind to the allosteric site of the enzyme, away from the active site. A competitive inhibitor does not change the three dimensional (secondary and tertiary structure) of an enzyme, but a non-competitive inhibitor always changes its structure. A competitive inhibitor will not affect the active site of the enzyme, but a non-competitive inhibitor will alter the active site of the enzyme such that it is no longer complementary in size, shape, and charge to the substrate. The effects of competitive inhibition can be overcome but increasing substrate concentration, thus increasing the chance of forming enzyme-substrate complex but the effects of non-competitive inhibition cannot be overcome by increasing substrate concentration, but only by increasing enzyme concentration. The effects of competitive inhibition are usually temporary but the effects of the non-competitive inhibition are permanent.

Q: Describe how globular proteins are synthesized after mRNA have been formed

There are three stages of translation. First, in initiation, a small subunit of ribosome binds to the mRNA transcript. Then, each aminoacyl-tRNA synthetase catalyses teh coupling of aminoacyl-tRNA to its specific amino acid. The first tRNA molecule, carrying methionine, occupies the peptidyl-tRNA site of the ribosome, alongside initiation factors. Initiation factors then dissociate from the small ribosomal subunit and make way for the large ribosomal subunit. For prokaryotes, small ribosomal subunit binds to its recognition sequence. The large ribosomal subunit joins the initiator complex. In the second phase, elongation, the second amino acid tRNA complex occupies the aminoacyl-tRNA site of the ribosome. Peptidyl transferase catalyses the formation of a peptide bond between teh two amino acids and the dipeptide is on the tRNA in the A site. Ribosome then translocates by a codon in the 5' to 3' direction. tRNA present in the A site is now translocated to the P-site and the uncharge tRNA in teh P site enteres and E site and is released. In the final stage, termination, the ribosome encounteres the stop codon (UAA, UAG, or UGA). Release factors bind directly to the stop codon mRNA and ribosome dissociates from one another, thus releasing the newly synthesized polypeptide.

Advantages of mitosis

Genetic stability - in the absence of mutations, there is no genetic variation as the parent is identical to the daughter as DNA replication ensures close to 100% fidelity with negligible error rate.
Growth - organisms can create new cells for growth or repair damaged cells
Asexual reproduction - it allows organisms such as Amoeba to divide by asexual reproduction.

Q: Explain why the two strands of DNA elongate in opposite directions

The two strands of DNA are anti parallel. Their 5' to 3' direction is in opposite direction. As the active site of the enzyme DNA polymerase III only recognizes the 3' end of a DNA nucleotide, free nucleotides can only be added in the 3' direction. Hence each of the two strands of DNA can only elongate in different directions when undergoing replication.

Q: Compare and contrast DNA replication and transcription

Similarities:
Both processes occur in the nucleus. Both processes also involve specific complementary base pairing. Both processes involving the unwinding of the double helix DNA. Both processes involve forming of hydrogen bonds between the original DNA and the eventual product.

Differences:
Replication involves the forming of a new DNA molecule but the product of transcription is an mRNA molecule. Replication uses the base thymine but transcription uses the base uracil. Replication uses deoxyribose sugar but transcription uses ribose sugar. Replication involves DNA polymerase whereas transcription involves RNA polymerase. In replication, the DNA does not remain intact as both parent strands are eventually separated under semi-conservative replication whereas in transcription, the DNA eventually remains intact. Replication occurs in order for mitosis to occur, it is part of the cell division cycle; however, transcription occurs as a part of protein synthesis.

Q: Compare and contrast transcription and translation

Transcription occurs in the nucleus but translation occurs in the cytoplasm and rough endoplasmic reticulum. The two processes use different components and enzymes, transcription uses transcription factors and RNA polymerase whereas translation uses ribosomes and aminoacyl tRNA synthetase. Transcription uses DNA as a template whereas translation uses mRNA codons as a template. Transcription results in an mRNA strand being formed whereas translation results in a polypeptide being formed. The building blocks (monomers) are also different; transcription uses RNA nucleotides whereas translation uses amino acids. Transcription involves the pairing of complementary bases of DNA and free RNA nucleotides whereas translation involves the pairing of anticodon bases of tRNA and those of mRNA.

Translation

Translation is the process by which a triplet base sequence of mRNA molecules are converted into a specific sequence of amino acids in a polypeptide chain in the cytoplasm of a cell. It consists of initiation, elongation, and termination.

The mRNA, a tRNA carrying the first amino acid of the polypeptide chain and the two subunits of a ribosome are brought together. After leaving the nucleus, the mRNA forms a reversible attachment to the mRNA binding site at the surface of a small ribosomal subunit. It recognizes a specific sequence on the mRNA just upstream of the initiation codon.
The initiation codon on the mRNA attracts its complementary tRNA, which carries the specific amino acid. This aminoacyl tRNA complex binds reversibly to the mRNA at the initiation site by hydrogen bonding via specific complementary base pairing with its anticodon.
It occupies the P site of a ribosome; translation starts as a translation initiation complex is formed. The initiation codon is most often AUG, which codes for the amino acid methionine.
Several ribosomes attracted to the mRNA simultaneously form a polyribosome. This is possible as once a ribosome has moved past the start codon, a second ribosome can attach to mRNA, thereby increasing efficiency of translation. The binding of the mRNA forms a translational complex and elongation of the polypeptide chain as translation proceeds. Newly formed polypeptide chains then enter the rough endoplasmic reticulum lumen and fold into its secondary/tertiary/quaternary structure in the cisternae. It is then packaged into vesicles and buds off and fuses with the soft endoplasmic reticulum and then the Golgi apparatus.

Amino Acid activation

Codons of an mRNA molecule contain genetic messages that are carried by the mRNA and they need to be translated to form the corresponding sequence of amino acids that will form the polypeptide chain and subsequently the protein.
The tRNA transfers amino acids from the cytoplasm's pool of amino acids. Subsequently, the translation is done by tRNA. Amino acid activation occurs when each amino acid attaches itself to a tRNA. It is very specific, as each tRNA can only be linked to one particular amino acid.
Each tRNA carries an anti-codon which is the cause of the tRNA's specificity. One specific tRNA recognizes and carries only the amino acid specific by the mRNA codon. Amino acid activation si catalysed by the enzyme amino acyl tRNA synthetase. There is only one AATS for every amino acid present in the protein. ATP is required in the process and amino acyl tRNA adenylate is formed.
One amino acid and one ATP molecule will attach to the enzyme's active site. The appropriate tRNA then binds to the active site of the enzyme AATS.
A covalent bond is then formed between amino acids and the 3' end of the tRNA, and ATP is hydrolysed. An activated amino acid (amino acyl tRNA is formed and released from the active site of the enzyme).

mRNA splicing

Genes of eukaryotic cells containing introns and exons. Introns, short for intervening sequences, are non-coding regions of a gene. They are transcribed but not translated into the amino acid sequence of proteins. Exons, or expressed sequences, are coding regions of a gene and they are transcribed and translated into the amino acid sequence of proteins.
During transcription, both introns and exons are transcribed from the DNA. The primary mRNA transcript therefore contains both introns and exons.
mRNA splicing occurs and special enzymes attach to mRNA strand and the introns are cut out and the two ends of adjacent exons are then connected together. The functional mRNA molecule that is exported from the nucleus is therefore much shorter and this is the RNA that will be translated to a protein molecule.

Transcription

Each gene contains a specific nucleotide sequence called the promoter region and it contains the initiation site, where proteins, called transcription factors, and RNA polymerase enzyme recognize and bind to. The enzyme-DNA-protein complex then causes the double helical DNA to unwind from the protective histones and H-bonds between bases break. The DNA in that region separates into its two polynucleotide chains and exposes the sequence of DNA complexes to be copied.
The enzyme then moves along the sequence of DNA until it reaches the initiation site. Translation is initiated and RNA polymerase catalyses the formation of a complementary RNA polynucleotide using the DNA sequence of the template strand as a template. It also ensures that the addition of RNA nucleotides occurs in a 5’ to 3’ direction, systematically, according to complementary base pairing. Hydrogen bonds then form between the base pairs. A and U, C and G, A and T. It also catalyses the formation of phosphodiester bonds when the free RNA nucleotides are added to the 3’ end of the growing nucleotide chain. Upon reaching the end of the gene, it reads the termination sequence and H-bonds between the base pairs will break. The RNA molecule is released from the DNA. Hydrogen bonds then form between the base pairs of the two DNA polynucleotide chains and it rewinds around the protective histones into a double helix again.