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BIOL1997 > BIOL1997 > Flashcards

BIOL1997 Flashcards

Module 1

1
Q

How is genetic material passed on through generations?

A

Cell division

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2
Q

How much DNA do humans share and where are the biggest differences in that DNA?

A
  1. 9%

- Individual’s immune system

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3
Q

What is the criteria for life?

A

o Homeostasis-maintaining a consistent internal environment
o Organization- being structurally composed of 1 or more cells
 Compartmentalization
 Cell specialization
o Metabolism-transformation of energy
o Growth- maintenance of a higher rate of anabolism than catabolism. A growing organism increases in size in all of its parts, rather than simply accumulating matter.
o Adaptation- the ability to change over time in response to the environment
 May adapt in evolutionary or behavioral sense
o Response to stimuli
o Reproduction

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4
Q

Describe common classification criteria

A

6 kingdoms:

  • Eubacteria
  • Archae
  • Protists
  • Plants
  • Fungi
  • Animals

3 domains:

  • Bacteria
  • Archaea
  • Eukarya
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5
Q

What are prokaryotes?

A

Lack membrane bound organelles

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6
Q

What are eukaryotes?

A

Have membrane bound organelles

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7
Q

What is an atom and what does it consist of?

A

• Atom- the smallest part of an element that can exist and retain the properties of an element
o Consist of nucleus with protons and neutrons
o Electrons which are waves existing in orbitals around the nucleus

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8
Q

How do electrons work?

A

 The further an electron orbital is from the nucleus, the greater is the energy of the electrons
 Electrons move from one orbital to another. Move up require a quantum of energy. Move down  release a quantum of energy.

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9
Q

What is atomic number?

A

Number of protons in nucleus

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10
Q

What is mass number?

A

Protons+neutrons

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11
Q

How are chemical properties of an element determined?

A

By the outermost electron shell

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12
Q

What elements is life based on?

A 
•	Carbon
•	Hydrogen
•	Nitrogen
•	Oxygen
•	Phosphorous
•	Sulfur
Nearly all living organisms are composed almost entirely of 16-18 elements, with a few other elements restricted to particular groups. H, O,N,C make up 99% of the living parts of the organism
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13
Q

What are the 4 molecules of life?

A

Molecules of life (all based on carbon):
• Nucleic acids (DNA/RNA)
o DNA more stable and easier to replicate heredity material from than RNA
• Proteins
• Fat/Lipids
o Cell membranes made of this to contain and concentrate molecules
• Sugars/Carbohydrates

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14
Q

What are useful properties of carbon to life?

A

• Life depends on carbon
• All major biopolymers have a substantially carbon backbone
• Atomic number 6
o 1s22s22p2
o Valence of 4-tetravalent
o Tetrahedral in shape
 Can form isomers
o Mid-range electronegativity
• C-C and C-H bonds are strong and unreactive
o Provides solid scaffolds
o However not unreactive enough that they won’t respond to sufficient change
• C can bond-
o To itself
o To metals
o To heteroatoms
• Geometrically flexible
o Capable of catenation
 When forming a single bond with itself, carbon atoms can rotate relative to each other which makes biomolecules flexible
o Forms chains, rings, multiple bonds
• Carbon compounds are relatively inert or kinetically stable to hydrolysis and oxidation
• In general, organic reactions tend to be under kinetic control (how quickly a reaction occurs) rather than thermodynamic control
o Means they are often favorable but slow and good targets for enzymatic control

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15
Q

What is a biopolymer?

A

a polymeric substance occurring in living organisms

• All linear biopolymers have a defined beginning and end

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16
Q

How are biopolymers synthesised?

A
  • Biopolymers are synthesized in one direction only increasing the backbone due to their different beginnings and ends and the chemistry surrounding that
  • Some of the monomer is lost in polymerization, leaving a “residue” incorporated in the growing chain
  • Biopolymer synthesis relies on dehydration reactions and are anabolic (requires energy
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17
Q

Draw the functional groups-

  • Alcohol
  • Ketone
  • Aldehyde
  • Carboxylic acid
  • Ester
  • Amide
  • Ether
  • Amine
A

Look at book

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18
Q

Does DNA change from cell to cell?

A

No. Instead, each cell uses a subset of expressed genes to achieve its structure and function(s)

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19
Q

What kind of chromosome do most bacteria and archea have?

A

A circular chromosome

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20
Q

What are features of eukaryotic genomes?

A

o Tend to be bigger
o Have linear chromosomes that contain centromere in the middle, at the end or off-centre of the chromosome
 End of chromosome is telomere
o DNA condensed into chromatin and wrapped around histone proteins

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21
Q

Describe the human genome

A
  • Linear
  • 6 billion (6x109) base pairs
  • Divided into 22 pairs of chromosomes (plus the sex chromosomes)
  • About 20,000 proteins can be produced
  • Often only one pair of copies of each gene (one on each chromosome pair= alleles)
  • Other genomes can be much smaller or much bigger
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22
Q

What is the central dogma?

A

DNA–> RNA –> Protein

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23
Q

What is the genome?

A

DNA

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24
Q

What is the transcriptome?

A

RNA

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25
Q

What is the proteome?

A

Protein

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26
Q

What is RNA and what are some properties of RNA?

A

o RNA is a polymer made of nucleotide monomers
o Self-replicating
o They can store information in the sequence order of their different nucleotide monomers
o Possess a wide range of catalytic activities capable of providing metabolic function
o RNA polymers can improve and adapt to changed or new environments

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27
Q

What are different kinds of RNA?

A 
	Micro RNA and small nuclear RNA
•	Used to regulate gene expression
	Ribosomal RNA
	Messenger RNA
	Transfer RNA
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28
Q

What are examples of what is included in the proteome?

A 
	Micro RNA and small nuclear RNA
•	Used to regulate gene expression
	Ribosomal RNA
	Messenger RNA
	Transfer RNA
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29
Q

What is the history of genetic inheritance discovery?

A

o Genetic inheritance understood before it was known what genes were made up of
o Nucleic acids seemed too simple, and hard to really purify from proteins so lots of controversy about which carried genetic information

o 1869- Miescher discovers DNA

o 1900s-DNA passed from generation to generation and linked to diseases

o Levene 1910- there is RNA/DNA and their chemical components
 Tetranucleotide hypothesis- components of RNA/DNA
 Thought the RNA/DNA were just single molecules floating around in the cell
 Thought different species had same amount of ATCG

o 1948- Chargaff’s rules:
 A=T and C=G and A+G=C+T
 Species variation of DNA composition

o	1953- Structure of DNA
	Rosalind Franklin
•	X ray crystallography 
•	Phosphate on the outside 
	Maurice Wilkins 
	James Watson and Francis Crick
•	Took data from Franklin and figured out the structure of DNA
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30
Q

What experiments were done to demonstrate with Streptococcus pneumoniae how DNA is passed on?

A

 1928- Griffith transformed the bacteria Streptococcus pneumoniae(experiment seen below)
-Injected Strain R (rough non-virulent), strain S (smooth virulent), heat killed smooth virulent and rough non-virulent+ heat killed smooth virulent into mouse

Results:

  • Strain R: healthy
  • Strain S: dead
  • Heat killed strain S: healthy
  • Heat killed strain S+ Strain R= Dead
  • —There was live strain S bacteria in blood sample from dead mouse

• Suggested that bacteria are capable of transferring genetic information through a process known as transformation- didn’t know what the transforming principle was

 1944- Avery, MacCleod and McCarty purified the bacteria until they got the ‘transforming principle’, and proved that this was the thing that was causing the bacteria to transform- identified as DNA
• Prepared protein-free, purified DNA from S bacteria after they had been killed by heat
• Non-capsule forming R-strain bacteria were exposed to the DNA extracts and a proportion was found to be transformed into the capsule forming virulent type
• These changes were permanent and inherited by the daughter cells
• Demonstrated that DNA was the carrier of genetic information

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31
Q

Describe the formation and experiment behind the one enzyme/one gene hypothesis

A

• 1941- Beadle and Tatum generated a series of strains of N.crassa, each of which carried a mutation in a single gene that was required to make one or other of these compounds
• These strains could, therefore, grow only when that particular compound was provided in the medium
• Mutations in different genes that led to a requirement for the same vitamin or amino acid were found to be blocked at different enzymatic steps in the biosynthesis of that compound.
• By this approach, a different gene was found to be necessary for each enzymatically controlled reaction required for the synthesis of the various compounds
• Later changed to the one gene-one polypeptide hypothesis
o Although enzymes are nearly always proteins, many proteins are not enzymes

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32
Q

Describe electrostatic interactions

A

• Electrostatic interactions can be weak or strong
o Ions- atoms have completely lost or gained and electron=charged
o Like to be around polar molecules and balances by opposite charge
o Electrostatic attraction between oppositely charged ions
o Can be strong or weak depending on environment

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33
Q

What are hydrogen bonds?

A

o Attraction between partially charged atoms involving hydrogen (electropositive)
o FON
o Moderate

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34
Q

What are Van der Waals interactions?

A

o Attraction between partially charged atoms (permanent or temporary): several types
o Individually weak, but lots together can be strong

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35
Q

What does hydrophobic mean?

A

• Hydrophobic-doesn’t have a set-up of partial or dipole-dipole charge
o Equal sharing of electrons
o Don’t like being around water and polar molecules
o Tendency of hydrophobic molecules to stick together and avoid water
o Can be strong or weak depending on environment

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36
Q

What does hydrophilic mean?

A

o Atoms have permanent unequal sharing of electrons making partial charges
o Don’t like being around water and polar molecules

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37
Q

What does aromatic mean?

A 
o	Conjugated double bond
o	There are flat structure, meaning you can stick up aromatics on top of each other
o	Cyclic (ring-shaped) flat molecules with conjugated (alternating single and) double bonds 
o	Usually hydrophobic
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38
Q

What is a covalent bond?

A

o Shared electrons between bonded atoms

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39
Q

What are nucleic acids?

A

A class of molecules found in all living cells

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40
Q

Describe the structure of nucleic acids

A

• Phosphates carry a negative charge to the backbone
• Nitrogen bases are bound to the sugars
–N-glycosidic bond links base and sugar
• 5’end- phosphate at the start of the polymer
• 3’end hydroxyl end-end of polymers
• Phosphodiester bond- high energy bond that links subunits
• Nucleic acid strand has a directionality

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41
Q

Describe the sugar-phosphate backbone and its properties

A

o Common, no matter what base is attached
 Slightly different in RNA and DNA but mostly the same
o Negative charge (phosphates)
o Hydrophilic (sugars and phosphates)

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42
Q

What are applications of the properties of the sugar-phosphate backbone?

A

 Electrophoresis
• Nucleic acids migrate in an electric field because they are charged. The distance they migrate depends on size
 Ethanol precipitation
• Nucleic acids become insoluble when mixed with salt (to neutralize charge) and ethanol

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43
Q

What is a nucleotide?

A

Base+sugar+phosphate

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44
Q

Besides being the building units of nucleic acids, what can nucleotides do?

A

 Besides being the building units of nucleic acids, these nucleotides, in the form of triphosphate esters (ATP) are the cellular currency of energy that drives chemical and physical processes in cells
 Other nucleotides, such as GTP, regulate proteins by causing conformational changes that can activate some or inhibit others

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45
Q

What is a nucleoside?

A

Base+sugar

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46
Q

What are the pyrimidine bases?

A
  • Thymine
  • Cytosine
  • Uracil
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47
Q

What are the purine bases?

A
  • Adenine

- Guanine

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48
Q

What is the difference between DNA and RNA in terms of hydroxyl groups?

A
  • RNA-ribose-hydroxyl group at the 2’ carbon

* DNA-deoxyribose- one less hydroxyl group at the 2’ carbon

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49
Q

What is the structure of nucleobases

A

Aromatic ring structures:

-Flat and planar

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50
Q

What wavelength do nucleobases absorb and why is this useful to know?

A

260 nm
o Can monitor purity by checking ratios of absorbance values for likely contaminating molecules
o A260:A280 (proteins)
o A260:A230 (carbohydrates/phenol)

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51
Q

What is the difference in pyrimidine and purine aromatic bases?

A

o Pyrimidines single ring aromatic base

o Purines double ring aromatic base

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52
Q

What is the difference in strength between GC and AT?

A

o C and G complement each other
 3 hydrogen bonds so stronger binding
o A and T/U complement each other
 2 hydrogen bonds so a bit weaker

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53
Q

What is Tm and when does it increase?

A

• Melting temperature, Tm
o The temperature when 50% of the 2 strands come apart
 Increases with increasing %(G+C)
 Increases with length

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54
Q

What are properties of the DNA double helix (B-DNA)?

A

• Double stranded-
o Provides two copies and a template for repair
o Obvious mechanisms for replication/transcription via base-pairing
• Stable-
o Not prone to degradation
o Cells can repair cytosine deamination
• Strands run in opposite directions
• Flat bases stack on top of each other (reduced A260) in middle of structure
o Aromatic
o Try to exclude water from middle of helix- hydrophobic
• Negative phosphates repel each other
o Electrostatic repulsion
• Right handed double helix
• Major and minor grooves
o The four bases seen from the grooves have different properties
o Backbone narrow on one side, wide on the other

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55
Q

What base can degrade into another, and why is this a problem in DNA?

A
  • Bases are generally stable except for the spontaneous deamination which turns C–> U (water goes in, ammonia goes out)
  • Occurs ~100 per day per cell
  • Not a good idea for DNA (stable genetic information); uracil in DNA is recognized as wrong and repaired
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56
Q

What are RNA base pairs?

A

CG and AU

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57
Q

What structure makes the RNA less stable?

A

• But the extra OH group on the sugar stops it forming B-DNA-type helices
o Also makes RNA susceptible to degradation

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58
Q

Why can tRNA be formed?

A

• Can form complex structures by hydrogen bonding with complementary bases elsewhere in the molecule, enabling RNA, to varying degrees, to fold back on itself and form stemloop structures

o This pairing results in the formation of four double-stranded regions (stem) interspersed among single stranded regions (loops)
o One of these loops contain the anticodon sequence while other loops function in binding to the ribosome, where translation takes place

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59
Q

What do amino acid sequences determine for proteins?

A

Structure, which determines function

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60
Q

What do proteins do?

A

• Proteins give the cell its shape, they form receptors, enzymes, hormones and growth factors, toxins, transporters and antibodies

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61
Q

Draw an alpha amino acid and describe electron exchange

A

Ionic form that predominates at pH7

A molecule containing an amino group (H2N) and a carboxylic acid group (OH-C=O) that are separated by one carbon, called the α-carbon (diagram in book)

NH2 normally gains an electron (at ph 7.4) while the COOH normally loses an electron (at pH of the cell)

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62
Q

What is pH and what affects pH?

A
  • pH refers to the concentration of hydrogen ions/protons pH= -log10[H+/H3O+]
  • Acids decrease the pH
  • Bases increase the pH
  • Most organisms maintain pH in a narrow range
  • Buffers maintain a constant pH (carbonate ion)
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63
Q

How are peptide bonds formed?

A
  • Two amino acids combine by condensation polymerization to form a dipeptide
  • Very energetically unfavorable- doesn’t occur spontaneously
  • Coupling of amino acids occurs with the loss of a molecule of water and is therefore called a dehydration reaction
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64
Q

What are amino acids joined by peptide bonds referred to as?

A

Residues

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65
Q

What is a polypeptide chain?

A

A large number of amino acids linked together via peptide bonds(H-N-C=O or H-N=C-O as sometimes electrons get mixed)

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66
Q

What is at either end of an amino acid chain?

A

• There is always a free amino group at one end of the chain, called the N-terminus, and a free carboxyl group at the other end, the C-terminus

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67
Q

What are some properties of the peptide bond?

A

• Partial double bond makes the peptide bond flat and rigid
• Partial charges (positively charged nitrogen vs negatively charged oxygen) encourage hydrogen bonding –> hydrogen bond donors= nitrogen, hydrogen bond acceptors= oxygen
o Solubility -try to draw this
• Can still rotate around other bonds
—alpha carbons: sidechains attach here: rotation around bonds either side

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68
Q

What is the charge of the amino acid C terminus?

A

-

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69
Q

What is the charge of the amino acid N terminus?

A

+

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70
Q

What are the two sulfurs in polypeptide backbone and what do they do?

A

o Aliphatic one behaves like a carbon

o Second sulfur can take up one form or another in redox reactions

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71
Q

What are the different properties of amino acid chains?

A
  • Size/shape
  • Charge
  • Polarity
  • –Polar non-ionic side chains with -OH, -SH or -amide
  • Hydrogen bonding potential
  • Hydrophobicity
  • -Hydrophobic aliphatic (chains of -CH2-)
  • Aromatic
  • Redox sensitive
  • Flexibility
  • Acidity or basicity
  • -Acidic: side chains with -ester
  • -Basic: side chains with -N+
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72
Q

What is the absorbance of protein?

A

280 nm

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73
Q

What is the effect of pH on amino acid chains?

A

o Above pH7 or so the amino acid chain will be unchanged
o Below pH7 the amino acid chain will probably experience change
• At pH5 the overall charge is ~0
• At pH8 the overall charge is ~-1

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74
Q

What is a primary protein structure?

A

 Amino acid sequence
 Order in which the amino acid units in a protein are joined together ultimately determines the way in which a protein will fold into its functional 3D structure

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75
Q

What is a secondary protein structure?

A

 Local structures
• Allow formation of structure
• Backbone-backbone hydrogen bonding interactions are very important
o Backbone carbonyl groups and amide nitrogen groups hydrogen and bonding patterns and bond angles help define the structure
• Sidechain interactions help hold the structure together and form the tertiary structure

  • Alpha helix
  • Beta sheet
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76
Q

Describe alpha helix and its sidechains

A

o Always have same basic shape, right handed helix
o Sidechains point outwards
o Sidechains can make interactions with other parts of the protein to form tertiary structures

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77
Q

Why is the A-helix perfect to fit into the major groove of DNA?

A

 Diameter of helix is on average 1.2 nm
 Major groove of B-DNA is 1.2 nm wide
 A common feature of DNA binding
 Sidechains must point the right way to recognize either the backbone for general/non-sequence specific binding, or the bases for sequence specific binding

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78
Q

Describe the beta sheet

A

o Can be parallel (strands point in the same directions) or antiparallel (strands point in opposite directions)
o Arrow points in direction of protein chains (N–>C)
o Side chains point above and below
o Sidechains can make interactions with other parts of the protein to form tertiary structures
o Beta turn- form of protein secondary structure, often formed between beta strands in beta sheet.

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79
Q

What is tertiary structure?

A

Overall 3D arrangement of polypeptide chain

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80
Q

Does folding of polypeptide chains occur spontaneously?

A

Folding can occur spontaneously, but most proteins are too large and require the assistance of a group of folding catalysts called molecular chaperones

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81
Q

How are proteins folded?

A

 Held together by lots of different interactions/bonds
• Hydrogen bonds
• Ionic/electrostatic interactions
• Hydrophobic interactions
 A driving force for protein folding is the hydrophobic effect
• Hydrophobic sidechains clump together on inside of the molecules
 pH, solvents and temperature are really important to maintain structure
 All the properties influence how the proteins are folded
1. Information encoded in the amino acid sequence
2. Burial of hydrophobic surfaces/sidechains in aqueous solvent
3. Collapse of protein chain/formation of secondary structure
4. Firming up tertiary structures by interactions between different parts of the protein

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82
Q

What is quartenary protein folding?

A

 Organization of subunits (many but not all proteins have multiple subunits)

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83
Q

In terms of structures, what structures do proteins have?

A

o Every protein has primary structure, most have secondary, some have tertiary, 2/3 of proteins have quartenary structure

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84
Q

Are biomolecules dynamic or fixed?

A
  • Proteins and other structured biomolecules are not rigid but “breathe” as atoms move around/bonds twist and lengthen/shorten within limits
  • They’re dynamic
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85
Q

Under what conditions does most of life on earth occur and why?

A

o 5 degrees to 50 degrees
o Atmospheric pressure
o Nearly neutral pH

  • Most biological macromolecules (proteins, nucleic acids) are unstable outside a narrow range of environmental conditions
  • Proteins can be hydrolyzed (broken down into their constituent amino acid residues) in very acidic or basic conditions with added heat/pressure
  • Proteins are much more easily unfolded- lose their unique 3 dimensional shape if heated
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86
Q

What does kinetic mean?

A

how quickly an event happens- related to the rate of reaction

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87
Q

What is a kinetic product?

A

the product that is formed faster

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88
Q

When is a reaction under kinetic control?

A

o When the temperature is low, the product ratio is determined by the reaction rate and such a reaction is said to be under kinetic control

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89
Q

What does thermodynamics mean?

A

measures the transitions of intrinsic energy- involves the final energy states

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90
Q

What is a thermodynamic product?

A

The product that is more stable

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91
Q

When is a reaction under thermodynamic control?

A

o When temperature is high, the reaction to form products is reversible; in this case the ratio of products is determined by the relative thermodynamic stabilities: such a reaction is said to be under thermodynamic control

92
Q

What will the substrate- product equilibrium be?

A
  • Favourable (give out energy- exergonic)

- Unfavourable (need energy input- endogonic)

93
Q

What is equilibrium?

A

rates of forward and reverse reactions are the same; concentrations of substrates and products don’t change; overall energies are balanced

o At equilibrium, the total free energy difference, ΔG, is zero

94
Q

Under standard conditions, does the substrate molecule or the product molecule have more free energy?

A

Substrate molecule

95
Q

What is the second law of thermodynamics?

A

o A process will only happen spontaneously (eg without added energy) if it increases the entropy of the universe as a whole

96
Q

What is Gibbs free energy?

A

o A measure of the amount of usable energy (energy that can do work) in that system

97
Q

What is the formula for change in Gibbs free energy?

A

o ΔG=Gfinal-Ginitial
o ΔG=ΔH-TΔS
 Where ΔH is change of enthalpy
• Enthalpy refers to energy stored in bonds. Change in enthalpy is the difference in bond energies between the products and reactants
 Where ΔS is change in entropy
• If positive: system becomes more disordered (eg when a large molecule splits into several smaller ones)
 Where T is temperature
• All biological reactions are temperature sensitive

98
Q

When is a reaction spontaneous?

A

• When ΔG<0, energy is released: spontaneous or exergonic

99
Q

When is a reaction non-spontaneous?

A

• When ΔG>0 , energy input is required: non-spontaneous or endergonic

100
Q

Can reactions be coupled? Why would you do this?

A

• Endergonic and exergonic reactions can be coupled so that one reactions supplies energy to another, transforming a non-spontaneous reaction into a spontaneous one

101
Q

What is metabolism?

A

represents the complete set of reactions that are involved in the degradation of fuel molecules to provide the cell with energy

102
Q

What is energy used for?

A
  • At a cellular level, energy is used to produce the biomolecules that form the fabric of the cell, to transport ions and molecules across membranes, to produce organized cell movement and to remove the waste products of chemical reactions and other toxic substances
  • Energy is also needed in the constant battle to maintain cell in their ordered, functional state.
103
Q

What is energy?

A

The capacity to do work

104
Q

What is free energy?

A

The amount of available energy from a reaction

105
Q

What is potential energy?

A

the energy stored and is the energy usually involved in biological systems

106
Q

What is chemical energy?

A

potential energy that is stored in the covalent bonds between atoms in molecules

107
Q

What is kinetic energy?

A

energy expressed as movement

108
Q

What is heat energy?

A

kinetic energy of randomly moving molecules

109
Q

What is radiant energy?

A

energy of any form of electromagnetic radiation

110
Q

What is the most common energy conversion in organisms?

A

Potential –> Kinetic energy

111
Q

What is activation energy?

A

energy required to initate a reaction

112
Q

In order to do work, what must reactions be?

A

Out of equilibrium

• The most useful reactions for providing energy are those with equilibrium constants lower than 1.0.

113
Q

What do catalysts do?

A

Reduce the activation energy of the reactants

114
Q

What are most biological catalysts? How do they work?

A

• Biological catalysts are enzymes:
o Most enzymes are proteins
o Enzymes bind to reactants in a very specific manner
o Binding to reactants lowers the activation energy by placing stress on chemical bonds
o Chemical reactions are sped up and more energy is released

Enzyme +Substrate –> Enzyme+ Product

115
Q

What is transition state?

A

High energy state that has to be bypassed to form products: when substrates are ready to react

116
Q

How are enzymes folded?

A

o Enzymes are generally globular proteins
o The polypeptide chains are folded in such a way as to form one or more pockets or grooves on the surface, creating a specialized region into which the substrate molecules can fit- this is known as the active site

117
Q

What are substrate-binding amino acids?

A

Amino acids that form an active site

118
Q

How do amino acids form the active site and let the enzyme do its thing? How do active sites work?

A

o They may be adjacent to one another in the polypeptide chain or on different parts of the polypeptide chain that are brought together by folding to give the tertiary structure of the enzyme
o The exposed side chains or R-groups of the amino acids line the active site and their detailed arrangement in relation to one another determines the specificity of binding of the enzyme to its substrate
o Some of the R-groups lining the active site are concerned in the specific binding and orientation of the substrate molecules
o The R-groups form charged, or uncharged, hydrophilic or hydrophobic surfaces in the active site, which bind precisely with particular parts of the substrate molecules
o Other active-site amino acids are involved with the making or breaking of covalent bonds- these are the catalytic amino acids. Substrate may form a transient covalent intermediate with a catalytic amino acid in the active site
o For the enzyme to perform its catalytic function, it is imperative that the substrate undergoing the chemical change is positioned precisely in relation to the catalytic amino acids, so that the rearrangement of atoms in the chemical reaction can occur: it is the role of the binding amino acids to direct the substrate to that position.
o After they use, catalytic amino acids regain their original structure and formula

119
Q

What is the induced fit model?

A

o Enzymes are specific-
o Relatively big, highly functionalized active sites
o Specific to their substrate
o Induced fit model-
 Enzyme changes shape/conformation as it binds substrate and stabilizes transition state

120
Q

What do enzymes vary in?

A 
o	Tend to classify enzymes with an ending of “-ase”, the beginning being what they do 
o	Mostly proteins (some enzymes are RNAs)
o	Highly varied in terms of:
	Function 
	Size 
	Need for co-factors
•	Metals
•	Other small molecules
	Ability to be regulated
121
Q

What are enzymes partly regulated by?

A

o Enzymes are partly regulated by compartmentalization. They can be:
 Inside or outside cells
 In particular cellular compartments
o Enzymes in the wrong place can be a sign of a problem or can cause a problem
 Cardiac enzymes in the blood stream (LDH)

122
Q

What do enzymes do to rate of reaction?

A

Increase

123
Q

Are enzymes used up in reactions?

A

No

124
Q

Are enzymes highly specific?

A

Yes

125
Q

Can enzymes be regulated?

A

Yes

126
Q

Can enzymes be organised into pathways?

A

Yes

127
Q

Can enzyme mutations cause disease?

A

Yes

128
Q

What is used for all enzyme kinetics measurements?

A
  • The initial linear rate is used for all enzyme kinetics measurements, but then it goes into a plateau
  • Change in concentration of product with time
129
Q

Under the same experimental conditions and high substrate concentration, doubling the enzyme will do what?

A

Double the rate

130
Q

What do DNA and RNA polymerase both do?

A

• Makes a copy of DNA or an RNA copy of DNA from a DNA template
• Always copy the new strand 5’ to 3’
• Can only work in one direction
• Uses nucleotide triphosphates as substrate
• Adds the nucleotide monophosphate to the 3’ OH end of the growing chain
• Requires a template
• Forms a phosphodiester bond
• Releases pyrophosphate (PPi)
o Release by hydrolysis of NTPs into NMPs; spontaneously forms phosphates (2Pi); provides energy for unfavorable reactions

131
Q

What is a primer?

A

Short sequence, usually of RNA, that pairs with a strand of DNA and provides a starting point (free 3’-OH end) for DNA polymerase to begin synthesis of the new DNA chain

132
Q

What are the differences between DNA polymerase and RNA polymerase?

A

DNA polymerase-

Need a primer to start

Proofread the last nucleotide added; 3’ to 5’ exonuclease activity

Use deoxynucleotide triphosphates (dNTPs, dATP, dGTP, dTTP, dCTP) as substrate

Makes a single new template for the next generation of cell

RNA polymerase-

Don’t need a primer to start

Don’t efficiently proofread the last nucleotide added; no 3’ to 5’ exonuclease activity

Use ribonucleotide triphosphates (NTPs: ATP, GTP, CTP, UTP) as substrate

Many copies transcribed, used, degraded

133
Q

What is reverse transcriptase, and why is it done?

A
  • There are DNA polymerases which make DNA from an RNA template; reverse transcriptase
  • Made by retroviruses (which have an RNA genome)
  • Used in molecular biology to make stable DNA copies of mRNA
134
Q

How are metabolic reactions organised

A

Into regulated metabolic pathways

135
Q

How is short term control or enzyme rate achieved?

A

 Modify the structure and therefore the activity of the enzyme polypeptide (covalent modification) , such as phosphorylation of polypeptides
 Feedback inhibition- where the end product of a metabolic pathway can bind to one of the enzymes involved in its synthesis
 Change the amount of an enzyme in the cell

136
Q

How does feedback inhibition work?

A
  • When the product of a pathway exceeds the amount that is required by the cell, this product binds to the regulatory enzyme in the pathway to shut it down until the level of product falls
  • Enzyme is often an allosteric enzyme; an enzyme that has both an active site where the substrate binds and another site where the end product binds
  • The binding of the regulatory molecule to the regulatory subunit causes it to change shape, which is transmitted to the catalytic subunit, causing it to change shape and interfering with the binding of the substrate and thereby the activity of the enzyme
137
Q

What does changing the amount of an enzyme in the cell involve?

A

• Involves the cooperation of transcription factors which bind to the regulatory region of a gene, causing the gene to be transcribed into messenger RNA encoding the protein

138
Q

What type of replication is DNA replication?

A

Semiconservative

139
Q

Do DNA strands run parallel or antiparallel to each other?

A

antiparallel

140
Q

How are dNTPs added during DNA replication?

A

o Building blocks are the deoxyribonucleoside triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP.
o Synthesis of DNA is always in a 5’ to 3’ direction relative to the strand being synthesized.
o Deoxyribonucleotides are added to the 3’ end of the DNA strand by the enzyme catalyzed formation of an ester linkage between the 5’ phosphate of the incoming dNTP and the 3’ hydroxyl (OH) of the end of the strand
o This forms a phosphodiester bond between the adjacent sugars, extends the sugar-phosphate backbone and leaves the 3’OH group of the newly added deoxyribonucleotide free for the addition of the next deoxyribonucleotide.
o During this process, two of the three phosphates of the dNTP are released in a covalently linked form known as pyrophosphate
 Hydrolysis of PPi provides energy
o Importantly, nucleotides are incorporated into the growing chain according to the rules of base pairing, so that the base added to the new strand is complementary to the base in the template: adenine opposite thymine and guanine opposite cytosine

141
Q

What is the experimental evidence by Meselson and Stahl that DNA replication is semiconservative?

A
  1. Bacteria were grown in 15N for several generations so that all of the DNA strands were more dense than normal
  2. The bacterial DNA was then allowed to replicate once or twice in normal 14N so that there were one or two rounds of new strand synthesis
  3. Growth in either 14N or 15N produces DNA of different densities, which can be separated by centrifugation in caesium chloride
    • Results:
    o The density after one round of replication was intermediate between the heavy and normal DNA, indicating that the original heavy DNA was no longer present but had been replicated in either a semiconservative or dispersive way, ruling out the conservative replication hypothesis
    o Following the second round or replication in 14N, DNA was found to be either intermediate between the heavy and normal, or the same as normal DNA: this result confirmed that DNA replication is semiconservative
142
Q

What does DNA lygase do?

A

Enzyme that joins the 3’ hydroxyl and 5’ phosphate ends of breaks in DNA strands by catalyzing the reformation of a phosphodiester linkage

143
Q

What does DNA polymerase do?

A

Enzyme catalyzing the addition of nucleotides to a growing strand of DNA in a 5’ to 3’ direction

144
Q

What does DNA gyrase do?

A

Enzyme that makes a transient cut in one strand of the DNA, allowing the supercoils and twists to be unwound before it accurately rejoins the phosphodiester bond it initially cut

145
Q

What does endonuclease do?

A

Enzyme that cuts DNA internally by hydrolyzing phosphodiester bond

146
Q

What does exonuclease do?

A

Enzyme that cuts DNA by removing bases sequentially from the ends of DNA strands by hydrolyzing terminal phosphodiester bonds

147
Q

What does the clamp loader do?

A

Pentamer that binds ATP and catalyses its hydrolysis to form ADP and phosphate
When the clamp loader binds to ATP, the loader opens

148
Q

What does the clamp protein do?

A

Hexamer that forms a closed donut- can slide freely among the DNA. When bound to an open clamp loader, it can interact with open DNA. This interaction leads to the hydrolysis of the ATP bound to the clamp loader and the closing of the clamp around the DNA
Clamp loader is released, and the DNA-clamp protein complex can interact with DNA polymerase and slide along the DNA- to remove the clamp, reverse the process
The clamp holds the DNA on the core polymerase of DNA pol III, increasing the processivity of the enzyme- ensures that DNA pol III doesn’t dissociate with the DNA

149
Q

What is the leading strand?

A

DNA strand that is synthesized continuously in a 5’ to 3’ direction

150
Q

What is the lagging strand?

A

DNA strand that grows in an overall 3’ to 5’ direction but is synthesized discontinuously in short fragments (5’ to 3’) that are later joined by DNA ligase

151
Q

What are the origin and terminus’ of DNA?

A

Sequences of DNA at which replication is initiated and terminated, respectively

152
Q

What is a replication fork?

A

Points of separation of strands of duplex DNA where replication occurs

153
Q

Talk about the origin of replication in bacterial chromosomes

A

• Bacterial chromosomes are circular and have a single origin of replication
o Initiation sites are usually AT-rich
o This is because there is less hydrogen bonding in AT than GC, so there is less work to pull the hydrogen bonds apart

154
Q

How does DNA replication occur? (focusing on bacteria but relevant for eukaryotes too with a few differences)

A
  1. Replication is initiated by a cut or nick (a single break of bond between adjacent nucleotides) in at least one strand, made by DNA gyrase
    a. Precise DNA sequences of origins contain recognition sites for specific DNA-binding proteins
  2. As the DNA unwinds with helicase, a replication fork is generated at each side of the origin by the initiation machinery
    a. Pulling long, helical strands apart causes supercoiling: topoisomerase enzymes cut strands, allow to unwind and religate them- they stop supercoiling
  3. Chain elongation- New strand assembly takes place at these replication forks as the double helix unwinds bit by bit
    a. Replication forks move at a constant rate
    b. Each replication fork contains a leading strand growing towards the fork and a lagging strand growing away from the fork
  4. DNA binding proteins prevent the strands from re-annealing together
    a. Single stranded-binding proteins coat single stranded DNA to keep strands apart- stop small segments of base pairing (which would cause hairpins) and protect DNA
  5. The two forks generated at the origin proceed bidirectionally around the circular bacterial chromosome, both strands being copied at the same time, and eventually meet at the terminus, where synthesis stops (the replication forks can also hit each other)
    a. Like the origin, the terminus of bacterial chromosomes has a specific sequence for the binding of terminator proteins
155
Q

How does chain elongation occur in the DNA replication process? (again focusing on bacteria but relevant for eukaryotes as well)

A

• DNA polymerase can only start synthesis by attaching a nucleotide to a free 3’OH group at the end of a pre-existing strand
o Polymerase is good at making phosphodiester bond

  • To initiate synthesis, then, a short strand of RNA with a 3’ OH terminus is added to the single-strand DNA template
  • Short strand is called a primer and is synthesized by primase
  • The RNA primer created by the primase then allows DNA polymerase III to commence synthesis from the 3’ end of the primer
  • When it has finished its task, the primer is removed by an editing 5’ to 3’ exonuclease
  • For the leading strand, 5’ to 3’ synthesis occurs in the same direction as the movement of the replication fork, so a single primer initiates synthesis at the replication origin and then DNA polymerase III continues to add nucleotides sequentially and continuously , keeping pace with the replication fork
  • For the lagging strand, it is synthesized as a series of short fragments, each of which initiates close to the replication fork and proceeds away from the fork until it meets the previous fragment

• Lagging strand synthesis is discontinuous, with primase laying down a series of primers as the replication fork progresses
 This is because if the Okazaki fragments did not occur and the parental strand was left to unzip completely, then there would be a possibility for the DNA strand to fold back on itself, get twisted up

• DNA polymerase III adds nucleotides to the 5’ end of the primers to lengthen them away from the fork until the next fragment is encountered, creating Okazaki fragments
o Okazaki fragments are produced slower
o The Okazaki fragments are separated into loops- once a loop has been finished, it is released and the next section is held into the loop
 DNA loops for regulation or because of protein interactions

  • When DNA polymerase III arrives at the 5’ end of a previously synthesized Okazaki fragment, it stops and departs
  • The process then restarts at the site of the next primer, which has been synthesized close to the replication fork
  • The leading strand now consists of one unbroken chain, whereas the lagging strand is punctuated by regular stretches of short RNA primers
  • DNA polymerase I enters at this point and, by the action of its own 5’ to 3’ exonuclease, replaces each RNA primer with DNA
  • Ligase then joins adjacent fragments together
156
Q

How many origins of replication do eukaryotes have?

A

Many

157
Q

Are the replication forks uni or bidirectional?

A

Bidirectional

158
Q

Do eukaryotes have smaller or bigger okazaki fragments than prokaryotes?

A

Smaller

159
Q

When does replication occur in eukaryotes?

A

o Timed to replicate just before cell division
 Complexes form at each origin of replication (the inactive forms)
 They get activated at the same stage of the cell cycle

160
Q

What needs to be done before and after DNA replication in eukaryotes?

A

o Need to strip off nucleosomes (consisting of a length of DNA wrapped around histone) before replication and reform nucleosomes immediately afterwards

161
Q

In human cells, is replication of the leading and lagging strands performed by the same or different DNA polymerase?

A

Different

162
Q

How fast does the replication fork in eukaryotes move compared to prokaryotes?

A

o A replication fork in eukaryotes moves about 20 times more slowly than in prokaryotes due to size constraints

163
Q

What do eukaryotic chromosomes have to speed up total replication of the chromosome?

A

Replicons (unit regions of replication)

164
Q

In eukaryotes, when do replication forks stop?

A

o As replication forks extend bidirectionally from an origin, they continue to move until they eventually join with replication forks from adjacent replicons or until a replication fork approaches the end of a linear chromosome

165
Q

Are eukaryotic replicons initiated at the same time?

A

o Not all eukaryotic replicons are initiated at the same time- groups of approximately 50 replicons appear to commence independently of other groups, so that a chromosome can be broadly divided into early, mid or late replicating regions
o The time of replication appears to be at least partly due to whether the region contains active genes or not- active genes replicate early

166
Q

What is a telomere?

A

sequences at ends of linear eukaryotic chromosomes

167
Q

What is the problem that telomeres need to overcome?

A
  • Completion of replication at each end of linear chromosomes presents a special problem
  • Because DNA polymerases can only elongate from a 3’ –OH, the replication machinery builds the lagging strand by a backstitching mechanism
  • RNA primers provide 3’-OH at regular intervals along the lagging strand template
  • While the leading strand goes all the way to the end of the template, the lagging strand stops short from the end
  • When RNA primers are replaced, there is no 3’-OH at the end of the chromosome to prime the replacement of RNA with DNA
  • Because of this inability to replicate the end, chromosomes would progressively shorten at the end of each replication cycle
168
Q

How do telomeres overcome the problem of chromosome shortening?

A
  • This problem is overcome by the presence of the telomere, a terminal sequence consisting of repeated DNA sequences; in humans, there are many repeats of TTAGGG
  • These repeats are added to the ends of chromosomes by a special polymerase, termed telomerase.
  • Telomerase template is provided by an RNA molecule within the telomerase complex
  • Henceforth, an RNA primer is set at the complimentary added sequences by the telomerase, and the original end of the chromosome (not the stuff added by the telomerase) is replicated complimentarily by DNA polymerase alpha.
169
Q

What 2 stages does the central dogma involve?

A

o Transcription- synthesis of an RNA intermediate from a DNA template
o Translation- enzymatic process in which a ribosome moves along the RNA and catalyses the synthesis of a polypeptide

170
Q

What is the primary transcript?

A

The initial transcript produced by RNA polymerase prior to RNA processing to produce the mature mRNA, rRNA or tRNA

171
Q

What is mRNA?

A

The mature messenger RNA, containing one or more open reading frames that is translated by ribosomes to produce polypeptides

172
Q

What is an intron?

A

Sequences that are removed during processing of the primary transcript

173
Q

What is an exon?

A

Sequences that are joined together during processing to form the mature mRNA

174
Q

What is splicing?

A

The processing of a primary transcript to remove introns

175
Q

What are 5’ to 3’ untranslated sequences?

A

Sequences in the mRNA, located either side of the open reading frame, that does not encode a polypeptide

176
Q

What is tRNA?

A

The adaptor RNA molecule, 75 nucleotides in length, that contains an anticodon complementary to a codon in the mRNA

177
Q

What is aminoacyl-tRNA?

A

tRNA to which the appropriate amino acid has been covalently attached

178
Q

What is an operon?

A

Sequences in bacterial DNA that encode a primary transcript and contain the cis-regulatory sequences required for regulated expression of that transcript

179
Q

What is a constitutive gene?

A

A gene expressed constantly

180
Q

What is an open reading frame?

A

A sequence of codons that begins with the AUG initiator codon, proceeds through a series of amino-acid-encoding codons and finishes with a termination codon

181
Q

What is a ribosome?

A

The rRNA-protein complex that provides a scaffold for the assembly of mRNA, peptidyl tRNA and aminoacyl tRNA and catalyses peptide bond formation during protein synthesis

182
Q

What is an inducible gene?

A

A gene expressed only under certain conditions

183
Q

What is a promoter?

A

The site to which RNA polymerase binds to initiate transcription

184
Q

What is an operator?

A

A cis-acting sequence to which a transcriptional repressor binds

185
Q

What is a repressor?

A

A protein that binds to an operator and prevents transcription

186
Q

What is an enhancer?

A

A eukaryotic cis-acting regulatory sequence that controls expression of a gene, independent of its orientation or precise location with respect to the promotor for that gene

187
Q

What is a transcription factor?

A

A protein that binds to enhancer sequences and regulates transcription – proteins capable of recognizing a specific base sequence
Known as the sigma factor in bacteria

188
Q

How does transcription occur?

A
  1. RNA polymerase binds to a part of DNA called the promoter which sits just upstream (past the 5’ end of the primary transcript) and the DNA unzips over a short length, just in that part of the DNA that holds the gene to be used
    a. RNA polymerase is a multisubunit enzyme, that associates with the DNA-bound transcription factors
    b. The transcription factors facilitate the binding of RNA polymerase to the single stranded DNA- the DNA is “melted” in the local region
    c. The -10 region often has the sequence TATA which is easy to melt as only two hydrogen bonds (one less than GC)
    d. In bacteria, at -10 and -35 is the promoter region, known as the consensus sequence.
    e. +1 is the transcription start site
    f. These numbers are relative to the start site
    g. Initiation is slow
  2. Transcription-Transcription of the gene occurs controlled by the enzyme RNA polymerase, which starts transcribing downstream from the promotor sequence
    a. Only one strand of DNA is being transcribed but which strand is being copied can vary
    b. The DNA acts as a template and RNA nucleotides are assembled, forming a complementary single-stranded mRNA molecule
    c. The sequence of nucleotide bases on the mRNA molecule is the same as the DNA coding strand, except that it has a U instead of T
    d. The introns are transcribed along with the exons, resulting in a very long mRNA molecule.
    e. Local transcription bubble as the RNA polymerase complex moves along; one of the components in the complex unwinds the DNA
    i. Local transcription bubble is actually small compared to the size of the polymerase
    ii. Whole polymerase actually sits on outside and through the middle of the piece of DNA that it is copying
    f. The 2 strands of DNA reanneal once the RNA polymerase has passed by
    g. When the enzyme reaches a terminator site, it stops transcription
    i. Transcription uses a signal encoded in the transcribe RNA to stop
    ii. Intrinsic transcription termination:
  3. A section of G/C rich sequence forms a stable intra-strand loop, followed by an A/T rich sequence
    a. The G/C hairpin slows the RNA polymerase down, and due to the low strength of A/T binding, it just falls off
  4. The RNA dissociates from the DNA template and the RNA polymerase leaves the DNA template
    iii. Extrinsic transcription termination-
  5. A Rho protein binds to a C/G rich signal in the RNA, and uses helicase activity to travel up to and dissociate the DNA/RNA hybrid complex
    h. This process is very fasted- in bacteria ~50 nt per second
    i. This process occurs in the nucleus in eukaryotes and in the cytoplasm in prokaryotes
    i. In prokaryotes, ribosomes can bind and initiate translation while transcription is still in progress as there is no nucleus separating mRNA and ribosomes
  6. After transcription RNAs are processed/modified:
    a. Capping occurs to modify 5’ end (mRNA only)
    b. Polyadenylation: Add A nucleotides to 3’ end  “poly-A-tail” (mRNA only)
    c. Splicing: removal of introns from final RNA (in eukaryotes only) by spliceosome. Exons are stuck back together
189
Q

What are some challenges transcription faces?

A
  • Only small sections of the genome need to be transcribed
  • These sections often have to be copied thousands of times
  • Some sections are rarely copied in one cell but copied many times in another cell
  • Where to start?
  • Where to stop?
  • How to switch on/off/upregulate
190
Q

How can genes be expressed at different frequencies?

A

• Genes can be expressed at different frequencies (including at different times/conditions) by:
o Promotor strength
 DNA sequence optimized for strong or weak sigma factor/RNA polymerase binding
 Promotor strong  transcribed at high levels
 Promotor weak  transcribed at low levels
o Repressors
 A protein repressor binds- this blocks the binding of the sigma factor/ RNA polymerase complex
 No RNA polymerase binding  no transcription  no gene expression
 Repression can be relieved by small molecule binding and change its shape a bit so repressor can’t bind well with the promoter
o Accelerators
 Happens when sigma factor/ RNA polymerase complex doesn’t bind strongly/often
 Weak promoter, DNA sequence is very unlike the general consensus sequence, which would mean low gene expression for this gene
 A transcriptional activator (a protein) binds at a specific DNA sequence and laters the structure of the promoter so the transcription factor can now bind more frequently
 The binding of this activator is often under the control of a key metabolite such as cAMP. The concentration of that metabolite regulates the binding of the activator.

191
Q

What is the differences between prokaryotes and eukaryotes in transcription?

A

Prokaryotes:

  • Sigma factor
  • Activators/repressors

Eukaryotes:
More transcription factors
-set of general/basal transcription factors

Proteins to modify DNA accessibility
-Chromatin remodelers/ Histone modifiers

Cell-gene specific transcription factors

Enhancers (DNA elements) and complexes that cause DNA looping

192
Q

What types of RNA does protein translation use and what are they for?

A

• Uses three types of RNA:
o Messenger RNA (mRNA)-
 Contains template for protein synthesis/information about which amino acids to add in which order
o Transfer RNA (tRNA)-
 Matches the correct amino acids to the template
o Ribosomal RNA (rRNA)-
 Combines with proteins to form the machinery for protein synthesis/catalyses peptide bond

193
Q

What are some challenges of protein synthesis?

A
  • Need to convert a sequence of nucleotides to a sequence of amino acids
  • The need to have the correct order of amino acids
  • Peptide bond formation is very thermodynamically unfavourable
194
Q

How many amino acids do we have?

A

20

195
Q

What is a singlet?

A

• Singlet- 4 different bases and use only one position then you have 4 possible combinations: A,G,C or T

196
Q

What is a doublet?

A

• Doublet- 16 possible pairs

197
Q

What is a triplet?

A

• Triplet- 64 possible pairs

198
Q

Why do we say that the amino acid code is redundant?

A

• There will be redundancy in the code, i.e. some amino acids can have more than one code

199
Q

In eukaryotes, is mRNA monocistronic or polycistronic?

A

• In eukaryotes, each mRNA codes for a single polypeptide (monocistronic) whereas prokaryotic mRNAs may code for several proteins (polycistronic)

200
Q

Do we have overlapping, partially overlapping or non-overlapping code?

A

Non-overlapping code

201
Q

What is a codon?

A

The combination of 3 bases which codes for an amino acid

202
Q

What are the 3 stop codons that terminate protein synthesis?

A
  • UAA
  • UAG
  • UGA
203
Q

What is the start codon?

A

Methionine: AUG

o Methionine can appear either as a starter codon or a simple amino acid- dual role

204
Q

Why is the genetic code said to be degenerate?

A

• Some amino acids have multiple codons such as Leucine- the genetic code is said to be degenerate
o The degeneracy in the code is particularly evident in the third nucleotide of a codon, where changes often do not alter the amino acid that is specified.
o Codons that specify the same amino acid are synonymous codons

205
Q

Do amino acids always have multiple codons?

A

No, some have a single codon only

206
Q

What does the open reading frame consist of?

A

o ORF consists of a start codon followed by a series of codons that specify the sequence of amino acids and concludes with a termination codon

207
Q

What determines the open reading frame?

A

• Start codon AUG determines the reading frame AND the first amino acid
o Protein synthesis starts at the start codon and ends at the stop codon. Anything outside of that isn’t read.

208
Q

Does the stop codon encode an amino acid?

A

No

209
Q

Are tRNA’s specific? Why/why not?

A

o Each type of tRNA can be covalently attached to a single amino acid and has 3 ribonucleotide anticodon sequence complementary to the codon that specifies the amino acid attached to the tRNA on the opposite end
o Anticodon, responsible for codon recognition, is found at the end of a loop in each tRNA molecule
o Complementary base pairing between the codon and anticodon ensures that the correct amino acid is brought to the site of protein synthesis

210
Q

How are aminoacyl-tRNA’s made?

A
  • The adenine residue at the 3’ end of a tRNA molecule becomes covalently attached to the appropriate amino acid by aminoacylation, creating an aminoacyl-tRNA
  • Such reactions are catalyzed by at least 20 different aminoacyl-tRNA synthetases, one required for the attachment of each amino acid to its specific tRNA
  • Each aminoacyl-tRNA synthetase enzyme puts one amino acid on to the tRNA molecule, whose anticodon subsequently pairs with the codon specifying that amino acid
  • Different aminoacyl-tRNA synthetases for each tRNA/amino acid combination
211
Q

What is the purpose of Aa-tRNA synthetases?

A

o Catalyze the activation of amino acids- preps amino acid to get ready for high energy bonds
o Use ATP hydrolysis to get the energy to make a high energy bond
o Attach the correct amino acid to its matched tRNA
o Recognize the anticodon and other parts of the tRNA

212
Q

What is the issue with condensation polymerisation for dipeptide formation?

A

o This reaction is extremely thermodynamically unfavorable due to the large amount of water around
 Peptide bond formation causes the loss of a water molecule -> not favorable in aqueous molecule
o Hydrolysis is always favored over condensation in an aqueous environment

213
Q

How does the tRNA synthase work?

A
  1. Amino acid and ATP binds to the active site of the aa-tRNA synthetase
  2. AMP is covalently bound to the amino acid, thereby activating it, which is accompanied by release and breakdown of pyrophosphate
  3. The correct tRNA binds to the synthetase. The amino acid is covalently attached to the tRNA.
  4. AMP is displaced by tRNA, creating an aminoacyl tRNA
    a. Bond between amino acid and 3’-OH of tRNA effectively stores the energy from the phospho-diester bond
  5. Aminoacyl tRNA (the charged tRNA) is released from the enzyme
214
Q

What are ribosomes made of?

A

• Ribosomes are made up of a specific set of RNA molecules and proteins

215
Q

What do ribosomes do ?

A

• The ribosome provides a scaffold for the mRNA and aminoacyl-tRNAs to assemble and provides a reaction chamber for the catalysis of protein synthesis

216
Q

How are ribosomes formed?

A

• Like tRNAs, each rRNA folds upon itself to form short, double-stranded regions.
o The resultant stem-loop structures in each rRNA give a characteristic three-dimensional structure, which is important for interactions with ribosomal proteins
• When the large and small subunits combine, they undergo conformational changes, forming a groove in which the mRNA molecule sits
o Assembled ribosomes exist only when they are involved in the process of protein synthesis

217
Q

What does the small subunit of the ribosome do?

A

Binds mRNA

218
Q

What does the big subunit of ribosomes do?

A

Positions amino acids and has enough space for tRNA

219
Q

What is the P site of ribosomes?

A

Protein group

220
Q

What is the A site of ribosomes?

A

Acceptor site

221
Q

How is protein synthesis initiated?

A

• Small subunit of the ribosome binds mRNA and Met-tRNA
o Special Met-tRNA binds at the start codon modified Met (N-formyl-Met) in prokaryotes; special initiator tRNA in eukaryotes (methionine)
o A bit of extra sequence in the mRNA helps to identify the start codon- Met code at beginning slightly modified than one at the middle
• Large subunits of the ribosome binds to the small subunit and synthesis is ready to begin

222
Q

How is amino acid elongation achieved in protein synthesis?

A

• The formation of an intact ribosome establishes two tRNA binding sites
o One site, the A site, binds the incoming aminoacyl-tRNA, while the other site, the P site, accommodates the peptidyl tRNA (the tRNA covalently linked to the growing polypeptide being synthesized
o Upon association of the large ribosomal subunit with the pre-existing complex, the initiating methionyl-tRNA occupies the newly created P site, ready for the elongation reactions to begin
• AA-tRNA comes in guided by anticodon/codon matching; activated amino acids are positioned next to each other
• Elongation of a polypeptide is a repetitive process involving sequential addition of amino acids to the growing polypeptide chain
o Eloongation phase begins with synthesis of the first peptide bond between the carboxyl group of the first amino acid and the amino group of the second amino acid
o This goes on
o Polypeptide chain moves from 5’ end to 3’ end: the polypeptide chain grows from the N-terminus towards the C-terminus
• Peptidyl transferase enzyme in ribosome catalyses peptide bond formation using energy stored in the aa-tRNA bond; first Met bound to second amino acid
o Breaks bond between tRNA and amino acid:
 The peptide attached to the tRNA in the P site is transferred onto the single amino acid attached to the tRNA in the A site
 The new peptidyl-tRNA then moves to the P site on the ribosome (translocation reaction) , displacing the tRNA into the cytosol, where it can become recharged by its specific aminoacyl-tRNA synthetase
o No extra energy required to make peptide bonds -> the amino acids already prepped for peptide bonding
o Peptidyl transferase- enzyme component of the ribosome that transfers the activated amino acids from tRNA to the growing peptide chain
• First tRNA released, ribosome moves along to next codon on mRNA etc.
o Translocation reaction causes the ribosome to move three bases along the mRNA so that the new peptidyl-tRNA passes to the P site without the anticodon dissociating from the codon in the mRNA to which it is base paired
o This brings the next codon in the mRNA to the vacant A site so that it can receive the next aminoacyl-tRNA
o Extra energy from GTP hydrolysis used to get the correct aa-tRNA in, and move the ribosome
o 2-3 residues added per second
o No amino acid matches stop codon or the tRNA would get stuck in the ribosome

223
Q

How does protein synthesis termination occur?

A

• When stop codon is reached at the A site on the ribosome, no tRNA matches and transcription is stopped as it stalls the process because there is no tRNA that recognizes such stop codons
• Protein release factor binds
o Protein that binds the stop codon to terminate translation
o Specific termination factors then mediate the hydrolysis of the aminoacyl bond between the polypeptide and the final peptidyl-tRNA resident in the P site, releasing both the completed polypeptide and tRNA from the ribosome
• Peptidyl transferase adds water instead of an amino acid, releasing the polypeptide
• The machinery disassembles once P site is vacant, and the parts can be reused

224
Q

What are differences between prokaryotic and eukaryotic translation, and what are applications of these differences?

A

• Transcription/translation linked in bacteria not in eukaryotes
• Ribosomes have slightly different sizes/makeup but the same basic function
o Prokaryotic ribosomes are smaller than those of eukaryotes
• Initiation uses different mechanisms to find the start site and have different versions of Met
• Different protein factors to help things along
• Applications:
o More opportunity to control gene expression (e.g. mRNA degradation can prevent protein synthesis)
o Antibiotics can target bacteria and not eukaryotic translation because of differences in the molecules involved

225
Q

How is rate of mutation increased?

A
  • High energy radiation

- Chemical mutagens

226
Q

How does base substitution occur, what results and what is the effect of this result?

A

• Chemical similarity between certain bases occasionally causes wrong base to be inserted in a DNA strand
• Effect of a therefore changed amino acid depends on its position in the protein being produced and on function of the protein
• Most mutations make no difference at all (silent mutation) but some are serious, for example sickle cell
o This is due to the redundancy of the genetic code
o In active site of enzyme, makes a drastic difference
• Missense mutation- mutations that later a codon so that it now specifies another amino acid (leads to the production of an altered polypeptide)
• Nonsense mutation- change the sequence of a codon to a stop codon: lead to a premature termination of mutation

227
Q

What are frameshift mutations and their effect?

A
  • Consist of extra bases being added to or deleted from a strand of DNA
  • If a single base is deleted or inserted, the reading frame for the rest of the sequence of the gene can be changed which is serious

BIOL1997 (5 decks)

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