SU5.

SLIDE3

Central carbon=alpha carbon

Amino acids are chiral: 4 different groups attached

Thus asymmetric

Side chain gives the molecule its identity

NB!!! SU 1 Properties of Biomolecule

SLIDE4

Spectroscopic properties: aromatic rings absorb UV light

Structures of amino acids are on page 80 and 81 of GG-VII

SLIDE5:

Nonpolar: important in processes that drive protein folding

Polar uncharged: R groups form hydrogen bonds with water (amide+OH groups)

nucleophillic roles in enzyme reactions

soluble in water

Acidic: R group=carboxyl groups

This information helps one understand methods of protein purification, analysis

and identification

SLIDE7:

A peptide bond is a chemical bond formed between two molecules when the

carboxyl group of one molecule reacts with the amino group of the other molecule,
releasing a molecule of water (H2O).

Pic from: https://www.khanacademy.org/science/biology/macromolecules/proteins-

and-amino-acids/v/peptide-bond-formation
SLIDE8:

Amino acids are weak polyprotic acids (contain min of 2 dissociable hydrogens)

Groups don’t dissociate easily

Dissociation is pH dependent

Table 4.1 will always be provided

SLIDE10:

Low pH: acidic: increased [ H+]: PROTONATED

High pH: basic: increased OH , less [H+]: DEPROTONATED

SLIDE12;

Molecules are polar and asymmetric

Sense/direction

The order of building blocks specifies information

~ letters make up words

Information can inturn be extracted/ retrieved from the structure of molecules

For retrieval , you require recognition

Recognition-> interaction->physiological activity

Recognition mediated via weak forces.

Complementarity aspect

SLIDE15:

It was recognized that bases specify a.a sequences

Now the question of how many bases was needed?

Does it overlap?

Is it continuous?

MATH algorithms fav a triplet of bases

42= 16 arrangements . Too little to account for 20 a.a.

43=64 arrangenents. More than enough

Universal meaning codon assignments are the same throughout all organisms

SLIDE17:

Codons with pyrimidine as second base yields amino acids with hydrophobic chains

Codons with purine as second base yields amino acids with polar or charged amino
acids

Codons representing same/similar amino acid are similar in sequence

3rd base irrelevant

=3rd base degeneracy

Degeneracy protective against mutational disruption

SLIDE18:

See SU 4 slides 18-19 (secondary genetic code and activation of amino acid covered
there as well)

How is the a.a. matched with its tRNA

1. mRNA has codon

• Specifies amino acid sequence

2. Amino acid added at 3’OH of CCA end of tRNA

• Process is catalyzed by amino acyl tRNA synthetases (1 enzyme per amino acid)

• Each amino acyl tRNA synthetase loads its amino acid only onto a tRNA
designed to carry it.

• In turn tRNA through its anticodon will recognize unique specific sequences of
bases in the mRNA through complementary base pairing

Secondary genetic code: code by which each aminoacyl-tRNA synthetase matches

an amino acid to tRNAs

So that tRNAs can interact with codons specifying the amino acid

3.

Reaction serves to activate the amino acid so it can form a peptide bond
Bridges codon-amino acid information gap

Ensures that proper amino acid loaded onto tRNA. mRNA translated with fidelity.
information transfer is accurate

SLIDE19:

Protein synthesis dependent on codon-directed binding of proper aminoacyl tRNA

Ensures amino acid is aligned according to the mRNA undergoing translation

Codon-anticodon pairing achieved

Degeneracy (different codons yield a particular amino acid ) in the genetic code at the
3rd base

• Resolve by increasing specificity: 1 anticodon for each of those codons

• Fewer anticodons so that 1 anticodon recognizes more than 1 codon (yielding

the same amino acid)

Crick noted: some organisms have less than 61tRNA.

More play at 3rd base of codon and 1st base of anticodon

Bp less stringent

More than 1 codon for most amino acids

tRNAs of particular amino acid (whether the 1 amino acid is coded by 5 plus codons)
will have 1 synthetase

Bias in codon usage e.g. Codons specifying Leu. CUG used more than 48 000 times vs
UUA (6000 times)

Greater bias to use CUG

When mutations alter codon to nonsense codon protein synthesis is terminated

Incomplete polypeptide yielded

Not always deleterious. Can confer advantages

SLIDE20:

tRNA synthetase has to

1. recognize amino acids and

 2. discriminate the various tRNAs

No universal structural feature that allows AMINO ACYL SYNTHETATSES to recognize a

specific tRNA

Unique combination of sequence elements allows for recognition

• 1 base in the anticodon

• 1st free bp after 3’CCA

• 1 or more of the 3bp in the acceptor stem

2nd genetic code is an operational code

AMINO ACYL SYNTHETATSES have to recognize varying sequence and structural

features of the different tRNAs during aminoacyl tRNA synthesis

Pic shows identity elements within the tRNA that are recognized by its specific
aminoacyl-tRNA synthetase.

SLIDE21:

Synthetase enzyme relies on anticodon for selecting tRNA for loading

Altering anticodon specifies whether tRNA would be loaded or not and also with which
amino acid

Although Recognition features reside in the anticodon recognition is not limited to the
anticodon

e.g. alanyl-tRNAAla synthetase recognizes G:U bp in acceptor stem

Altering the bp abolishes aminoacylation with alanine

If pairing partner is U, amino group is free

Unpaired NH2 group at tRNA acceptor stem marks tRNA for acylation

Interaction between alanyl-tRNAAla synthetase and G:U bp causes conformational

change along the acceptor stem

3’CCA of stem enters enzymes active site and is aminoacylated

When G:U bp is lacking , 3’CCA end wont enter active site and is not aminocylated.

SLIDE22:

Class I and II synthetases

Class I : Add amino acid to 2’OH of A in CCA then shift to 3’OH (bind via minor groove)

Class II: Add amino acid to 3’OH directly (bind via major groove)

Formation of aminoacyl-adenylate

Transfer of activated amino acid moiety to 2’OH or 3’OH of ribose located on the 3’CCA
end of the tRNA

SLIDE23:

At equilibrium hydrolysis of peptide bond is favored

Slide 6. peptide bond formation requires energy

ATP used to drive reaction to be energetically favourable

Activates amino group by P it.

Slide 8: Flow of energy from sun onward

Energy release mediated by biomolecules

ATP = high energy molecule

ATP=P acceptor and donator

Stores energy for short times

High energy does not imply instability

Infact, require increase act energy to break bonds

Our concern: net release

ATPs free change in energy is negative

Sometimes reactions unfavorable

Couple to others so G=neg

SLIDE24:

COOH+amino groups of 2 amino acids interact

Covalent Amide bond forms

Water released

Hydrolysis is favoured at equilibrium

Couple peptide bond formation with energy input (ATP)

SLIDE25:

• Carbonyl oxygen and amide H2 usually found in the trans conformation- less
steric hindrance

• Energetically favorable

• If consider structure of the peptide bond you will notice

• The bond is characterized by single bond between carbonyl carbon and

amide nitrogen, see (a)

• Rotation is possible at any covalent bond i.e. at N-Cα, Cα-Co,and Co-N

because these are single bonds

• Co and O are linked via pie bond

• Nitogen left with lone pair of electrons

• Another resonance is possible i.e. Co and N participates in a pie bond

(Resonance describes delocalized electrons in a molecule)

• Lone pair now on oxygen, see (b)

• Co-N becomes double bond, restricts rotation

• Real nature of the peptide bond is between these two extremes

• Peptide bond said to have partial bond character, see ( c )

https://www.quora.com/What-is-a-partial-double-bond-What-causes-it

SLIDE26:

Consequences:

Restricted movement around peptide bond

Rotation still possible at : N-Cα and Cα-Co

Due to the double-bond character of the peptide bond, the six atoms of the peptide
bond group define a plane – the amide plane

SU6:
SLIDE3:

1. Flexibility in the sugar phosphate backbone

2. Glycosidic bond

3. Ladder = unfavourable conformation. Minimize interaction of water with bases

Therefore makes right handed twist-> minor /major grooves

4. A/B/Z forms

5. Denatured/Separate DNA fragments according to density

6. Coiling (tertiary) --→ Chromosomes (quaternary)

SLIDE4:

• The phenomenon

• How was it discovered experimentally ?

• Model developed?

• Transposons as examples of mobile DNA fragments -> genetic recombination

SLIDE5:

Non homologous recombination: occurs at low frequency ,

powerful force reshapes the genomes of organisms

Homologous recombination: prevalent during the production of gametes(meiosis)

Meiosis: a type of cell division that results in four daughter cells each with half the
number of chromosomes of the parent cell

Recombination in higher organisms can occur in the DNA of somatic cells (any cell of a
living organism other than the reproductive cells.)

Somatic cells are responsible for expressing proteins of the immune response

Somatic recombination: rearranges Ig genes increasing diversity of these molecules

from a fixed amount of genetic info.

Transposons :Also known as insertion sequences

Can cause mutation if a gene or regulatory protein at the site is disrupted

SLIDE7:

Breakage

• Denaturation/melt

• Melting temp: 50% DNA dissociated to single strands

Hybridization:

• piece of DNA /RNA of known nucleotide sequence identifies fragment with

complementary sequence

• reunion of strands needs to occur

How well hybridize/ re-associates/re-anneals:

• dependent on base composition :G:C more stable, higher

• Strand length: longer more bp

• Reaction conditions: high cation conc favours dsDNA

Similar sequences more for fixing DNA so information is not lost

Different/altered sequences for evolution

____________________________________________________________________________

Infect bacteria with 2 respective viruses

Viruses grown in 13C and 15N containing media (heavy and light)

Recover progeny by density centrifugation,

phage particles with recombinant genotypes distributed throughout the gradient bt

heavy and light

Parental particles as distinct heavy and light bands in the gradient

The recombinant phage had DNA in varying proportions from both parents

Dna must have “broke” and rejoined

Another observation: some plaques formed via single virus infecting a bacterium had 2
different genotypes

Some of the phages must have had a region of heteroduplex DNA to begin with

(a part of each strand is contributed by a different parent)

SLIDE8;
2 homologous DNA duplexes are juxtaposed( sequences aligned)

Chromosome pairing=synapsis

Single stranded nicks in the DNA at homologous sites of the 2 paired chromosomes

2 duplexes partially unwinds (RecBCD enzymes mark site for recombination

(NUCLEASE DOMAIN) + helicase function to unwind+repair DNA)

Single strand end of 1 duplex bp with nearly complementary ss region along intact
strand in the other duplex and vice versa

=strand invasion

Ligation of the free ends from the different duplexes

This yields cross stranded intermediate=HOLLIDAY JUNCTION

The junction migrates either direction (unwinding and rewinding of 2 duplexes)

Results in strand exchange

To resolve 2 duplexes: planar representation

Another pair of nicks introduced

1. patch recombination heteroduplex

- nicks in strands originally nicked. 1strand of duplex remains intact

2. Splice recombination heteroduplex

- Nicks in strands not previously nicked. 2 strands are heteroduplex

SLIDE9:

Barbara McClintock identified activator genes in maize → mutation of 2nd gene

Activator gene was an internal source of mutation

Activator and mutated gene in same DNA

Activator genes moved about freely in the genome

1983 she was awarded Nobel Prize in Physiology /Medicine

It was soon appreciated that Organisms possessed “jumping genes”

Genes which moved from 1 site to another in the genome--→ definition

Mobile/transposable elements/transposons
SLIDE10:

Transposons are segments of DNA enzymatically moved from 1 place in genome to

another

Their location within DNA is unstable

Range in size: several hundred bp to >80kbp

Transposons contain a gene which encodes an enzyme

1. for movement of the transposon to different locations and

2. insertion into chromosomes

Movement = transposition events

Transposons are also referred to as insertion sequences

Transposons can cause mutation if a gene or regulatory protein at that site is disrupted

DNA REPAIR VIA

1.HOMOLOGOUS RECOMBINATION,

2. EXCISION REPAIR AND

3. MISMATCH REPIAR

TRANSPOSITION: DRIVES EVOLUTION

SLIDE11:

Artificial gene rearrangement via experimental manipulation

Artificial DNA products= recombinant DNA

Use of recombinant DNA= genetic engineering

Nucleases digest/break down nucleic acids

Can act non-specifically or specifically

e.g. DNases act on DNA

RNases act on RNA

These enzymes cleave single or double stranded DNA

Functions of nucleases:

• DNA/RNA metabolism
 • Rearranging genetic info

• Host defense

• Immune responses

• catalysts

SLIDE12:

RESTRICT OR DEFEND AGAINST TAKEOVER BY FOREIGN DNA THAT MAY ENTER THEIR
CELLS

When sticky ends are formed:

Fragments have ss 5’ ends

Protruding ends have complementary base sequences

If fragments from DNA of different organisms combined, novel forms of DNA created

When different RE cleave identical sites=Isoschizomers

SLIDE15;

RE cuts DNA at unique sites

Yields fragments of diff sizes

Separate via molecular sieve and

Resolve complex mixture of fragments via electrophoresis

Sep according to size

Large molecules retarded.

Small molecules unhindered, migrate easily.

can be mapped/ordered according to size

SLIDE19:

Example of clone: 1 bacterial cell on petridish-→ colony of bacterial cells

Within transformed bacteria, recombinant DNA is carried as a plasmid

• The plasmid=clone of the original DNA molecule (plasmids

can be isolated+studied)
 If cloned molecule=gene (codes for a product), one can isolate and study the
product

SLIDE22:

RE cleaves circular plasmid, linearizes it

Linearized ends of plasmid joined to DNA fragment (if complementary ss overhangs)

CIRLCE CLOSES again

i.e. DNA ligase seals interruptions in the sugar P backbone---→ recombinant/chimeric

plasmid

Ligaase ccovalently links adjacent 3’OH and 5’ P groups

Ligase covalently links 3’OH and 5’ PO4 groups

HYBRID MOLECULES : plasmid DNA sequences + inserted DNA elements

Foreign sequences do not disrupt replication of plasmid

Recombination irrespective of DNA source

Limitation is size inserts into plasmid. Too large, plasmid not replicated effectively

Repeat sequences are difficult to clone e.g. telomeres, centromeres,

Bacteriophages manipulated so seq of 40kbp inserted in the bacteriophage genome

____________

insertion of sequences at random

Can also be directional

Insert gene coding for product into vector

Place downstream of promoter

Promoter= nucleotide sequence upstream to gene

Control expression of gene

RNA polymerase bind promoter and initiate transcription (DNA to RNA)

To get an insert with specific orientation , create DNA with different overhangs

Ligation in 1 direction=directional cloning.

SLIDE24:
Most often bacteria are transformed

Cells are made permeable with heat shock+Ca2+ treatment

Select for transformed cells

Small plasmids so insert size can be maxed!

Single ori. Time to complete replication depends on plasmid size

SLIDE27:

DNA Library=set of cloned fragments

Collectively represent the genes of a specific organism

Grow bacteria

Replica of the colonies on a disc

Treat disc with alkali to get ssDNA

Allow ssDNA to react with probe (defined sequence that is labelled)

Sequence complementary to the probe will be highlighted

Colonies with target DNA can be recovered from the plate.

SLIDE31:

Human genome project

Draft completed in 2000 and published in 2001

Bioinformatics: nature and organization of biological info

Genomics; addresses aspects of gene expression

Transcriptome: all genes expressed in a cell under defined set of conditions

Proteomics: protein expression

Human genome project imp in medicine.

Many diseases traced to occurrence of genetic defects

Make changes in DNA:

Commercial production of biomolecules e.g. insulin

Crops resistant to herbicides

Transgenic mice

Gene therapy

SLIDE32:

Repair damage due to genetic deficiency

Introduce functional version of defective gene

Gene incorporated into organism,

Expressed at selected time in selected cells

1. Isolate cells from patient

Co culture cells with vector that carries the functional gene

Reintroduce modified cells into host

OR

2. Directly introduce vector (carrying the gene) into host

e.g. retrovirus. Make retrovirus replication deficient

Will make DNA copy of RNA via RT

Integrate into host GENOME

Expression --→ gene product