Nucleic Acid 4

Question 1

Studying DNA :
can reversibly
denature —>

renature/anneal

Answer 1

1. H bonds & hydrophobic
   interactions are disrupted.
   DNA helix unwinds into 2
   strands (NO covalent
   bonds broken).
2. Is REVERSIBLE, esp if >10
   bases are still held
   together, unwound
   segments spontaneously
   rewind = REANNEAL

Question 2

When might ANNEALING OCCUR?

Answer 2

1. Can also have different DNA samples annealing, depending on their similarity in PRIMARY STRUCTURE (base sequence).

Question 3

hybridisation

Answer 3

DNA hybridization involves hybridizing the DNA from two different species based on complementary base pairing.

Question 4

Solutions of carefully isolated, native DNA are
VERY viscous at pH 7.0 at room temperature

Answer 4

1. Extremes of pH or elevated temperature cause denaturation
2. DECREASED VISCOSITY
3. Can determine %
   denaturation, by studying
   change in viscosity

Question 5

DNA Hypo/er-chromism = 3

Answer 5

1. Single-stranded (SS)
   DNA absorbs light more effectively than double-helical (DS) DNA
2. Can determine %
   denaturation of DNA
   by looking at change
   in A260.
3. As DNA denatures, get increased absorption
   = HYPERCHROMIC SHIFT

Question 6

DNA Denatures at Characteristic Temperature

Answer 6

1. Tm = melting
   temperature.
2. Temp at which
   DNA has reached
   HALF TOTAL MAXIMUM DENATURATION.
3. Can measure this by
   DECREASE IN VISCOSITY &
   INCREASE IN ‘UV’ LIGHT ABSORBED (HYPERCHROMIC EFFECT)

Question 7

Differences in Melting Temperature…

Answer 7

Tm variation between two different DNA preparations
reflects BASE COMPOSITION AND LENGTH OF DNA =

differences in forces holding the two sets of DNA strands together

Question 8

Which A, T,G, C compositions affect DNA melting Temps?

Answer 8

1. Recall A T pairs have
   2 H bonds,
2. whereas
   C G pairs have 3 H
   bonds, i.e. are more
   stable.

Hence, as GC
content increases, Tm
increases.

Question 9

Other Factors Affecting Melting Temperature :

Salt i.e. ionic strength

Answer 9

1. The concentration of ions in the solution will affect the stability of DNA
2. INCREASE in [salt] stabilising effect, why?
3. Ions such as Na+ will interact with the negative
   charges on the phosphate backbone of DNA, this
   reduces the repulsion between the negatively charged phosphates (major force in destabilising the double
   helix),
4. thus stabilising the duplex structure.
5. Hence:
   ——DNA in water denatures at room temp (20ºC)

——–DNA in 0.15 M salt denatures at T&raquo_space; 20ºC higher

Question 10

Other Factors Affecting Melting Temperature: pH

Answer 10

1. pH: The ionisation state of molecules depends on pH
2. DECREASE pH —-> PROTONISATION
3. INCREASE pH —> DEprotonisation.
4. CHANGES IN PROTONISATION STATE LEADS TO LOSS BETWEEN COMPLEMENTARY STRANDS. n state
   leads to loss of H bonds
   between complementary
   strands.

Question 11

when pH is lowered
to extremes (v acidic), can
cause

Answer 11

BREAKAGE OF GLYCOSIDIC BONDS.

So ALKALINE SOLUTIONS are preferred for DNA DENATURATION

Question 12

Characteristics of RNA = 2

Answer 12

1. RNA bases absorb
   at 260nm, but
2. as RNA has fewer long
   sequences of
   complementarity
   than DNA, it has
   FEWER BASE PAIRS &
   hence LOWER VISCOSITY… BROADER MELTING CURVE
   (similar to SSDNA).

Question 13

Other Factors Affecting Melting Temperature: SOLUTES IN VITRO

Answer 13

1. Solutes in vitro that can form H bonds lower Tm
   (decrease stability) of DNA double helix.
2. Organics like formamide & urea LOWER DENATURATION TEMPERATURE A& PREVENT REANEALING ON COOLING BY FORMING H BONDS with the bases and thus IMPEDING COMPLEMENTARY BASE PAIRING.
3. Note: need high concentrations to be effective

Question 14

Genes - functional units of DNA…

Answer 14

Organism = human

A human body is made up of trillions of cells.

each cell nucleus contains an identical complement of chromosomes in 2 copies. Each copy is a genome.

One specific chromosome pair.
- Each chromosome is one long DNA molecule, and genes are functional regions of this DNA.

DNA is a double helix.

Question 15

What is a Gene?

Answer 15

1. Before DNA structure was elucidated, genes defined as inherited factor/s determining characteristic/s
2. Vague definition – what a gene does but not what it is!
3. 1902: Archibald Garrod suggested that genes encode proteins.
4. Could we define a gene as a set of nucleotides
   that specifies amino acid sequence of a protein?
5. We now know this concept of a gene is a vast
   oversimplification…

Question 16

To fully understand DNA function (eukaryotes): 5

Answer 16

1.DNA replication

2. gene structure
3. gene transcription (making RNA, nucleus)
4. translation (protein synthesis, ribosomes, cytoplasm)
5. regulation of gene expression

Question 17

The central dogma summarises protein synthesis: 2

Answer 17

1. DNA is TRANSCRIBED into mRNA which is TRANSLATED into
   a polypeptide (protein).
2. The cell decodes the GENETIC CODE of a gene (the sequence of nt bases) to make a protein.

Question 18

1958: Watson & Crick
proposed that

GENES & PROTEINS ARE COLINEAR….EXPLAIN

Answer 18

1. Direct correspondence
   between nucleotide
   sequence of DNA &
   protein amino acid
   sequence.
2. Suggests that number of
   nts in a gene should be
   proportional to the
   number of amino acids in
   the protein encoded by
   that gene, but…

DNA: —-A CONTINUOUS SEQUENCE OF NUCLEOTIDES IN THE DNA….

TRANSCRIPTION

mRNA

TRANSLATION

POLYPEPTIDE CHAIN …..codes for Continuous sequence of AMINO ACIDS IN THE PROTEIN.

CONCLUSION: with colinearity, the number of nucleotides in the gene in proportional to the number of amino acids in the protein.

Question 19

Gene Structure (for a protein-encoding gene)

Answer 19

1. TRANSCRIBED REGION: contain information for the nt sequence of the gene product.
2. REGULATORY REGIONS
   contain sequences that are recognised & bound by
   proteins that transcribe the DNA into RNA.
3. PROMOTERS: Proteins also influence the amount of RNA made.
   Regions close to the transcriptional start site form the promoter
4. ENHANCERS: sequences far away are enhancers.

5.Many proteins associate with a gene prior to starting transcription

Question 20

DOWN STREAM VS UPSTREAM:

Answer 20

1. Not all of the DNA of a gene codes for an RNA:
2. a region immediately UPSTREAM (i.e., 5’ to the coding area) called the PROMOTER , is involved in INITIATION OF TRANSCRIPTION.
3. An area DOWNSTREAM (3’ to the coding
   region) is involved in TERMINATION OF TRANSCRIPTION.
4. INTRONS = spliced out to form mRNA
5. EXONS = encode polypeptide, EXPRESSED, included in mRNA

Question 21

There are two strands in DNA Consider a gene encoding a protein…

Answer 21

1. Coding
2. Template, Complement

3.By convention, always state sequence of DNA & RNA

4. 5’-3’. AND always refer to the CODING STRAND (since it has the same sequence & direction as the mRNA).
5. DS DNA——- TRANSCRIPTION —> mRNA.

Question 22

PROMOTER AND CODING STRAND:

Answer 22

1. The PROMOTER is named relative to the CODING
   STRAND on the DS DNA (same sequence as the
   RNA that is transcribed).
2. So, the promoter is 5’ to the Transcription start site on the gene - which is double stranded DNA…

Question 23

Gene Structure (summary): 5

Answer 23

1. The direction of a gene is always described relative to the CODING strand.
2. The transcription start site is labelled as nucleotide +1.
3. Nucleotides 5’ of this site are designated “upstream”,
   while nucleotides 3’ are “downstream”.
   “Upstream” nucleotides are labelled -1, -2, etc.
4. Therefore, the promoter is upstream & at nucleotides
   -10 to -30 (approximately in the diagram).
5. Coding region is downstream & at nucleotides +15 …

Question 24

Eukaryotic Genes consist of stretches of

“coding” & “non-coding” nucleotides (5)

Answer 24

1. Coding regions = exons & non coding regions = introns
2. All introns are initially transcribed into RNA, but later removed.
3. The exons are joined (spliced) to yield mature mRNA.
4. Introns common in eukaryotic genes but rare in bacterial genes
5. Size & number introns appears related to organism complexity
   (yeast short introns, Drosophila longer introns - up to a point!)

Question 25

A”Typical” Protein Encoding Gene

Answer 25

1. The gene’s two DNA strands are ANTIPARALLEL.
2. PROMOTER – DNA sequence to which TRANSCRIPTION
   APPARATUS BINDS TO INITIATE TRANSCRIPTION.
3. This indicates
   DIRECTION OF TRANSCRIPTION & WHICH OF THE 2 DNA STRANDS is the TEMPLATE STRAND.
4. TEMPLATE (ANTISENSE) strand is TRANSCRIBED INTO mRNA.
5. CODING (SENSE) strand – nt BASE SEQUENCE CORRESPONDS
   to RNA made, is the reference strand, 5’ – 3’
6. Coding region often contains: EXONS interrupted by
   INTRONS, intervening sequences between exons.
7. RNA POLYMERASE is the ENZYME THAT TRANSCRIBES THE RNA, USING COMPLEMENTARY BASE PAIRING WITH THE TEMPLATE STRAND TO GENERATE THE CORRECT RNA.