General structure for an amino acid.

The R group determines the chemical bonding ability of the amino acid - depending on the different R groups, the amino acid may be able to participate in hydrogen bonding, hydrophobic interactions, salt bonds, or disulphide bridging.

Proteins

Primary structure of proteins is held together by covalent peptide bonds.

Secondary structure (e.g. alpha helix, B-sheet, or random coil) is held together by hydrogen bonding.

Tertiary structure (the overall folded appearance of the protein) is held together by hydrogen bonding, hydrophobic interactions, salt bridges, and disulphide bridges.

Quaternary structure (the organization of multi-subunit proteins) is held together by hydrogen bonding, hydrophobic interactions, salt bridges and disulphide bridging.



illustration by Darryl Leja, National Human Genome Research Institute http://www.nhgri.nih.gov/DIR/VIP/

The amphipathic character of the lipids allows them to form membranes.  That is, these molecules have one polar end and one hydrophobic end.  Thus they self-assemble into layers with the hydrophobic portions together and the polar areas together. The hydrophobic portions want to be away from water while the polar portions want to be in contact with water. This results in a bilayer with a hydrophobic center.

The two types of nucleic acids are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). They are similar in that both are information carrying polymers (as opposed to structural polymers such as collagen), both use the bases A (adenosine), C (cytosine) and G (guanosine), both have backbone structures consisting of alternating units of 5 carbon sugars and phosphate, and both can be single or double stranded depending on the situation, and both are assembled by enzymes.

Differences include: DNA is usually double stranded, it uses T (thymine) as the fourth base, the sugar for the backbone is deoxyribose, and it stores genetic information (replication is more stringent than for RNA). RNA translates genetic information into structures such as proteins, uses U (uracil) as the fourth base, and the sugar for the backbone is ribose.

Phosphodiester bonds hold DNA together.

This is due to the limited space within the DNA double helix (where the bases sit) and the need to maximize hydrogen bonding potential (which in turn maximizes the stability of the DNA helix). Two purines (e.g. A and G) together would be too large to be accommodated in the available space (the sides of the helix would bulge). Two pyrimidines (e.g. C and T) would be too small (the bases wouldn't be within hydrogen bonding distance). Purine-pyrimidine pairs are the correct size for the observed diameter of the double helix.  A and T paired together are in the correct position to allow the formation of two hydrogen bond pairs, while C and G paired together allow the formation of three hydrogen bond pairs.

An AT base pair. Note the two hydrogen bonds holding the bases in place (hydrogen bonds are shown by dotted lines).
illustration from: www.ncc.gmu.edu/dna/two.htm



A GC base pair. The three hydrogen bonds holding the bases together make this a more sturdy pairing than the AT pair shown above (hydrogen bonds are shown as dotted lines). illustration from: www.ncc.gmu.edu/dna/three.htm

The basic chemical formula for a carbohydrate is (CH₂O)_n.

Six carbon sugars include glucose, fructose, galactose, etc.

A glycosidic bond is a bond formed between two sugar monomers by a dehydration reaction (with the loss of water).  The example shown above is a 1,4 glycosidic bond between two monomers of glucose.

Enzymes increase the rate of chemical reactions in the cell by lowering the activation energy necessary for the reaction to occur. Enzyme names usually end in the suffix "ase".  An example of an enzyme is amylase, a protein which breaks down starch.

Ionized molecules do not pass readily through the plasma membrane because it requires a very large amount of energy to move charged molecules through the hydrophobic core of the plasma membrane.  The energy needed to disrupt the hydrophobic bonds between the fatty acid tails of the phospholipids is not spontaneously available.


illustration from: http://www.accessexcellence.com/AB/GG/memPerm.html

Ribosomal RNA (rRNA) makes up the scaffolding for the ribosome.  All ribosomal proteins bind to either the rRNAs or to proteins bound to the rRNAs.

Messenger RNA (mRNA) carries instructions for building proteins from the DNA to the ribosomes.  Acts as a template for protein synthesis.

Transport RNA (tRNA) moves amino acids to the ribosome.  This folded molecule has two distinct ends. One end which attaches to the amino acid it carries; the other which is the anticodon which base pairs with the codon on the mRNA to ensure that the correct amino acid is added to the growing peptide chain.



illustration from: http://gened.emc.maricopa.edu/Bio/BIO181/BIOBK/tRNA2.gif

Three carbohydrate based structures in prokaryotes include: capsules, slime layers, lipopolysaccharide (outer membrane), peptidoglycan, storage granules of poly-β-hydroxybutyrate or glycogen.

DNA acts as a genetic information storage center.

Prokaryotic DNA is a single circular chromosome, and it is compacted by histone-like proteins.  Eukaryotic DNA consists of many linear chromosomes which are compacted by histones.

Prokaryotes do not undergo meiosis, they multiply by binary fission. During this process, their single chromosome becomes attached to the membrane at two points which move apart, ensuring that each daughter cell receives one copy of the chromosome. Note that prokaryotes are already in cells, and have no need for a reductional division to produce gametes. They do not reproduce sexually except to a limited extent (F plasmids), which rarely result in more than a partial diploid state for the recipient cells.

The organelles in question are the mitochondria and chloroplasts. Evidence for the endosymbiont theory includes:

1. Both organelles are approximately the same size as an average bacterium.
2. Both organelles reproduce by binary fission (as bacteria do).
3. Both organelles have their own closed circular DNA.
4. Both organelles have their own 70S ribosomes (same size and function as for bacterial ribosomes - eukaryotic ribosomes are larger - 80S) and their own tRNA.
5. There are currently examples of endosymbiotic relationships which may be at an earlier stage in their association (i.e. not yet as intimately involved as eukaryotic cells and their chloroplasts or mitochondria).

N-acetylglucosamine and N-acetylmuramic acid are the sugars which make up the peptidoglycan backbone.

Peptidoglycan determines the cell shape and supports the plasma membrane against osmotic pressure when the cell is growing in a hypotonic environment relative to the internal conditions in the cell.

Lysozyme is an enzyme which cuts the glycosidic bonds of the sugar chains of peptidoglycan between the N-acetylmuramic acid and the N-acetylglucosamine. This weakens the net-like structure of the peptidoglycan, and allows the cell to lyse due to osmotic pressure.

Penicillin is an antibiotic which acts a molecular mimic for the peptide portion of the peptidoglycan subunits. It binds irreversibly into the active site of the penicillin binding proteins (a family of proteins which are responsible for polymerizing peptidoglycan) and prevents peptidoglycan synthesis. It also binds to the active site of the transpeptidation enzymes which crosslink the peptidoglycan chains. This prevents the crosslinking of the chains and further weakens the peptidoglycan net. The penicillin also binds to the peptidoglycan hydrolases. These enzymes normally function to make small nicks in the peptidoglycan to allow the insertion of new subunits so that the cell wall can grow as the cell gets larger. Penicillin makes these enzymes more active, resulting in many nicks in the already established peptidoglycan, but there are no new subunits being inserted (Penicillin binding proteins are not functioning). Thus the peptidoglycan net becomes unstable, and eventually the cell lyses due to osmotic pressure.

Peptidoglycan is assembled in three distinct parts. In the first part, soluble enzymes link the N-acetylglucosamine (glcNAc) and N-acetylmuramic acid (murNAc). The peptide is then assembled enzymatically onto the murNAc part of the subunit. That is, each amino acid of the peptide is added by a separate enzyme, rather than having the entire peptide assembled on a ribosome and then attached to the disaccharide. The second part of the process involves moving the assembled subunits across the plasma membrane. The subunits are carried across the membrane by a carrier lipid (undecaprenol phosphate), and released on the other side of the membrane. The final part of polymerization involves the polymerization of the subunits into short chains and their insertion into the peptidoglycan net by the penicillin binding proteins. Once in place, the new chains are crosslinked to older parts of the net by the transpeptidases.

Peptidoglycan is a large net like structure in which individual chains are covalently linked to chains on either side or in the layers above and below. The extensive crosslinking between chains and layers makes this polymer strong and stretchy. It supports the cell envelope because it limits how far the plasma membrane can stretch due to osmotic pressure by providing mechanical support to the membrane.

You expect the Gram positive cell to retain the primary stain (purple) - the peptidoglycan will dehydrate during the selective decolourizing step (acetone alcohol rinse) which will trap the primary stain within the cell.

You expect Gram negative cells to be red. During the decolourizing step, the acetone alcohol will act as a dehydrating agent to shrink the pores in the peptidoglycan and as a lipid solvent to dissolve portions of the membranes and to mobilize the primary stain. Because the peptidoglycan is so thin in Gram negative cells as compared to Gram positive cells, the dehydrating action is unable to trap the dye, which is lost in Gram negative cells during this step. Thus, the Gram negative cells have many available binding sites for the secondary stain, which is red.

The outer membrane of Gram negative cells can be separated into an inner and outer leaflet. The outer leaflet is made up primarily of lipopolysaccharide,(a polymer unique to the outer membrane). The inner leaflet is made up mostly of phospholipids. The entire membrane contains many embedded proteins which are responsible for transporting or allowing movement of various substances across the outer membrane. Many of these proteins are part of the porin family.

A transport protein which moves two soluble substrates across a membrane at the same time in the same direction.  The energy required is generated by at least one of the substrates moving down its concentration gradient as it crosses the membrane.  The other substrate is usually being concentrated against its concentration gradient.

There are two main ways in which a protein can be membrane bound: it may have a hydrophobic anchor or it may have a hydrophobic surface.  If the protein has a hydrophobic surface, it may become bound into the structure of the membrane. In many cases, the protein will have a pore or channel in its center which is hydrophilic.  Alternatively, the protein may have a covalently attached fatty acid or phospholipid which will become associated with a membrane, and the protein then becomes membrane bound. If the hydrophobic anchor is removed, the protein usually is quite water soluble.

A porin is a hydrophobic barrel shaped protein with a hydrophilic channel passing through its center.  It is a membrane protein found in the outer membranes of Gram negative bacteria and mitochondria which allows the nonspecific passage of hydrophilic molecules through the membrane. All the molecules must be small enough to pass through the central pore and will be moving down their concentration gradient. This is a form of passive transport.

There are several options for moving molecules across membranes against their concentration gradients:

1. binding protein systems using ATP as the energy source (active transport).
   The solute and ATP will bind to the membrane transport protein, the ATP will be hydrolyzed, and the energy released will allow the transport of the solute across the membrane.
2. active transport using the proton motive force as the energy source.
   May use a symporter (direct use of the proton motive force) - the proton will enter the cell coming down its concentration gradient and this energy will drive the importation of the second solute.
   Alternatively, the proton motive force may be used to generate a second chemical gradient (e.g. Na⁺) which can then be harnessed in an antiporter to import the second solute while exporting Na⁺ ions.
3. group translocation: the substance being imported is modified during transport to maintain an apparent concentration gradient which is higher outside than inside the cell. For example, glucose can be imported into the cell down its concentration gradient (and therefore "under its own power") by phosphorylating it during transport. Thus a high concentration of glucose-phosphate is being generated inside the cell, but the concentration of glucose inside the cell is very low.  As a result, glucose will continue to drive its own entry into the cell.

The expected sequence is: UAC GGU ACA UAA CGC GUU

There are six codons in the sequence.

Prokaryotic and eukaryotic ribosomes are similar in function and form, but differ slightly in size.  Both are made up of a large and a small subunit.  The assembled prokaryotic ribosome is a 70S structure made up of a 30S and a 50S subunit.  The small subunit has a 16S rRNA and 21 proteins, while the large subunit has a 5S and a 23S rRNA as well as 34 proteins.

The eukaryotic ribosome is an 80S structure made up of a 40S small subunit (18S rRNA, 33 proteins) and a 60S large subunit (5S, 28S, and 5.8S rRNAs, 49 proteins).

1. initiation: mRNA binds to the small ribosomal subunit, and the initiator tRNA binds to the initiator codon.
2. the large ribosomal subunit binds to the above complex.
3. The ribosome has 2 active sites, the A (aminoacyl) site and the P (peptidyl)site. The initiator tRNA is in the P site.  An aminoacyl tRNA will recognize the mRNA codon which is present in the A site.
4. A peptide bond forms between the amino acids on the two tRNAs (the growing chain is transferred onto the tRNA in the A site).
5. The codons translocate one position down to the P site
6. Repeat until the stop codon arrives in the A site.

A tRNA (transfer RNA) is a single stranded RNA, which moves amino acids into the correct position on the ribosome for incorporation into growing proteins. These molecules are L-shaped when folded.  One end of the L is enzymatically linked to an amino acid, the other end is the anticodon that binds to the codon sequence of the mRNA.

illustration from: http://gened.emc.maricopa.edu/Bio/BIO181/BIOBK/tRNA2.gif

An RNA polymerase is an enzyme which combines ribonucleotides into an RNA chain. These enzymes are DNA dependent - that is, they require a DNA molecule to act as a template. These enzymes make all the types of RNA present in the cell.

illustration from: http://biotech.icmb.utexas.edu/glycolysis/pathway.html

You generate a net 2 ATP.

The final product is 2 molecules of pyruvate.

illustration from: http://chemistry.gsu.edu/glactone/PDB/Proteins/Krebs/Krebs.html

The products of the TCA cycle are 2 CO₂, 1 GTP, 3 NADH, 1FADH₂ per turn of the cycle. (Note: cycle turns twice for 1 glucose molecule entering glycolysis.)

The total per turn of the cycle is 1 ATP (from the GTP), 9 ATP (generated from the 3 NADH), and 2 ATP (generated from the FADH₂) for a grand total of 12 ATP per turn of the citric acid cycle.

This is the first part of the electron transport chain. The NADH reducing equivalents (2 electrons and 1 proton)and a proton from the bulk solvent in the cell are passed to the NADH coenzyme Q reductase. This enzyme complex will eject the protons to the intermembrane space and pass the electron to ubiquinon (a mobile electron carrier). The Ubiquinon will pass the electron to cytochrome reductase enzyme which will pick up a proton from inside the cell to balance the charge of the electron.  Cytochrome reductase will pass the electrons to cytochrome C and will eject the proton to the outside of the cell.

illustration from: http://gened.emc.maricopa.edu/Bio/BIO181/BIOBK/chemiosm1.gif

In the second part of the electron transport chain, cytochrome C will pass the electrons to cytochrome oxidase which picks up a proton from inside the cell. The enzyme splits oxygen and combines it with two protons. Another proton is ejected across the membrane in the process. This results in a build up of protons on the outside of the membrane, which causes a charge differential to form known as the proton motive force. This proton motive force can be used to generate ATP via the ATP synthase enzyme complex. Protons are allowed to flow down their concentration gradient into the cell, and the enzyme harnesses this energy to convert ADP and inorganic phosphate into ATP.

illustration from: http://gened.emc.maricopa.edu/Bio/BIO181/BIOBK/chemiosm2.gif

The purpose of the electron transport chain is to recycle the reducing equivalents (NADH,  and FADH₂) generated during glycolysis and Kreb's cycle to NAD⁺ and FAD, to create the proton motive force and conserve the bond energy of the sugar.

The cell can use this gradient to generate ATP, to move, to transport substrates (use the proton gradient, either directly or indirectly), etc.

Thirty eight molecules of ATP can be generated per glucose molecule by aerobic respiration.

No, because the alternative electron acceptors (e.g. nitrate, sulphate, etc) have a lower reduction potential than does oxygen. That is, the difference between the donor NADH and the inorganic acceptor molecule is less than between NADH and oxygen. The energy yield is related to the size of the reduction potential - a smaller energy yield allows less ATP to be made.

An anaerobic energy yielding process in which the final electron acceptor from the electron transport chain is an inorganic molecule other than oxygen.

Examples include beer, wine, cheese, antibiotics, amino acids, yogurt, sour cream, etc.

An energy yielding process in which organic molecules act as both the electron donor and the electron acceptor.
For example: glucose may be converted to pyruvate by glycolysis (glucose donates the electrons to NADH). Pyruvate can then act as the electron acceptor to regenerate NAD⁺ and pyruvate is converted to lactic acid.

Note: this is a relatively inefficient way to make energy, so for organisms which do not rely exclusively on fermentation, it will not occur in the presence of oxygen.

Three alternative electron acceptors for anaerobic respiration include nitrate, sulphate, carbon dioxide, elemental sulphur, and ferric iron.

A microorganism which does not require oxygen for growth, but that does grow better in its presence.

A microorganism that requires low levels of oxygen for growth, around 2 to 10%, but is damaged by normal atmospheric levels of oxygen.

A homolactic fermentation takes sugars and generates lactic acid as the sole fermentation product (e.g. lactic acid bacteria used for making yogurt).

A heterolactic fermentation takes sugars and generates a mixture of lactic acid, ethanol and CO₂.(e.g. some yeasts used in the brewing industry)

Examples of the biochemical products of mixed acid fermentations include ethanol, acetic acid, formic acid, lactic acid, succinic acid, carbon dioxide, hydrogen gas and butanediol.