Nucleic Acid 4 Flashcards
Studying DNA :
can reversibly
denature —>
renature/anneal
- H bonds & hydrophobic
interactions are disrupted.
DNA helix unwinds into 2
strands (NO covalent
bonds broken). - Is REVERSIBLE, esp if >10
bases are still held
together, unwound
segments spontaneously
rewind = REANNEAL
When might ANNEALING OCCUR?
- Can also have different DNA samples annealing, depending on their similarity in PRIMARY STRUCTURE (base sequence).
hybridisation
DNA hybridization involves hybridizing the DNA from two different species based on complementary base pairing.
Solutions of carefully isolated, native DNA are
VERY viscous at pH 7.0 at room temperature
- Extremes of pH or elevated temperature cause denaturation
- DECREASED VISCOSITY
- Can determine %
denaturation, by studying
change in viscosity
DNA Hypo/er-chromism = 3
- Single-stranded (SS)
DNA absorbs light more effectively than double-helical (DS) DNA - Can determine %
denaturation of DNA
by looking at change
in A260. - As DNA denatures, get increased absorption
= HYPERCHROMIC SHIFT
DNA Denatures at Characteristic Temperature
- Tm = melting
temperature. - Temp at which
DNA has reached
HALF TOTAL MAXIMUM DENATURATION. - Can measure this by
DECREASE IN VISCOSITY &
INCREASE IN ‘UV’ LIGHT ABSORBED (HYPERCHROMIC EFFECT)
Differences in Melting Temperature…
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
Which A, T,G, C compositions affect DNA melting Temps?
- Recall A T pairs have
2 H bonds, - whereas
C G pairs have 3 H
bonds, i.e. are more
stable.
Hence, as GC
content increases, Tm
increases.
Other Factors Affecting Melting Temperature :
Salt i.e. ionic strength
- The concentration of ions in the solution will affect the stability of DNA
- INCREASE in [salt] stabilising effect, why?
- 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), - thus stabilising the duplex structure.
- Hence:
——DNA in water denatures at room temp (20ºC)
——–DNA in 0.15 M salt denatures at T»_space; 20ºC higher
Other Factors Affecting Melting Temperature: pH
- pH: The ionisation state of molecules depends on pH
- DECREASE pH —-> PROTONISATION
- INCREASE pH —> DEprotonisation.
- CHANGES IN PROTONISATION STATE LEADS TO LOSS BETWEEN COMPLEMENTARY STRANDS. n state
leads to loss of H bonds
between complementary
strands.
when pH is lowered
to extremes (v acidic), can
cause
BREAKAGE OF GLYCOSIDIC BONDS.
So ALKALINE SOLUTIONS are preferred for DNA DENATURATION
Characteristics of RNA = 2
- RNA bases absorb
at 260nm, but - as RNA has fewer long
sequences of
complementarity
than DNA, it has
FEWER BASE PAIRS &
hence LOWER VISCOSITY… BROADER MELTING CURVE
(similar to SSDNA).
Other Factors Affecting Melting Temperature: SOLUTES IN VITRO
- Solutes in vitro that can form H bonds lower Tm
(decrease stability) of DNA double helix. - 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.
- Note: need high concentrations to be effective
Genes - functional units of DNA…
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.
What is a Gene?
- Before DNA structure was elucidated, genes defined as inherited factor/s determining characteristic/s
- Vague definition – what a gene does but not what it is!
- 1902: Archibald Garrod suggested that genes encode proteins.
- Could we define a gene as a set of nucleotides
that specifies amino acid sequence of a protein? - We now know this concept of a gene is a vast
oversimplification…
To fully understand DNA function (eukaryotes): 5
1.DNA replication
- gene structure
- gene transcription (making RNA, nucleus)
- translation (protein synthesis, ribosomes, cytoplasm)
- regulation of gene expression
The central dogma summarises protein synthesis: 2
- DNA is TRANSCRIBED into mRNA which is TRANSLATED into
a polypeptide (protein). - The cell decodes the GENETIC CODE of a gene (the sequence of nt bases) to make a protein.
1958: Watson & Crick
proposed that
GENES & PROTEINS ARE COLINEAR….EXPLAIN
- Direct correspondence
between nucleotide
sequence of DNA &
protein amino acid
sequence. - 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.
Gene Structure (for a protein-encoding gene)
- TRANSCRIBED REGION: contain information for the nt sequence of the gene product.
- REGULATORY REGIONS
contain sequences that are recognised & bound by
proteins that transcribe the DNA into RNA. - PROMOTERS: Proteins also influence the amount of RNA made.
Regions close to the transcriptional start site form the promoter - ENHANCERS: sequences far away are enhancers.
5.Many proteins associate with a gene prior to starting transcription
DOWN STREAM VS UPSTREAM:
- Not all of the DNA of a gene codes for an RNA:
- a region immediately UPSTREAM (i.e., 5’ to the coding area) called the PROMOTER , is involved in INITIATION OF TRANSCRIPTION.
- An area DOWNSTREAM (3’ to the coding
region) is involved in TERMINATION OF TRANSCRIPTION. - INTRONS = spliced out to form mRNA
- EXONS = encode polypeptide, EXPRESSED, included in mRNA
There are two strands in DNA Consider a gene encoding a protein…
- Coding
- Template, Complement
3.By convention, always state sequence of DNA & RNA
- 5’-3’. AND always refer to the CODING STRAND (since it has the same sequence & direction as the mRNA).
- DS DNA——- TRANSCRIPTION —> mRNA.
PROMOTER AND CODING STRAND:
- The PROMOTER is named relative to the CODING
STRAND on the DS DNA (same sequence as the
RNA that is transcribed). - So, the promoter is 5’ to the Transcription start site on the gene - which is double stranded DNA…
Gene Structure (summary): 5
- The direction of a gene is always described relative to the CODING strand.
- The transcription start site is labelled as nucleotide +1.
- Nucleotides 5’ of this site are designated “upstream”,
while nucleotides 3’ are “downstream”.
“Upstream” nucleotides are labelled -1, -2, etc. - Therefore, the promoter is upstream & at nucleotides
-10 to -30 (approximately in the diagram). - Coding region is downstream & at nucleotides +15 …
Eukaryotic Genes consist of stretches of
“coding” & “non-coding” nucleotides (5)
- Coding regions = exons & non coding regions = introns
- All introns are initially transcribed into RNA, but later removed.
- The exons are joined (spliced) to yield mature mRNA.
- Introns common in eukaryotic genes but rare in bacterial genes
- Size & number introns appears related to organism complexity
(yeast short introns, Drosophila longer introns - up to a point!)
A”Typical” Protein Encoding Gene
- The gene’s two DNA strands are ANTIPARALLEL.
- PROMOTER – DNA sequence to which TRANSCRIPTION
APPARATUS BINDS TO INITIATE TRANSCRIPTION. - This indicates
DIRECTION OF TRANSCRIPTION & WHICH OF THE 2 DNA STRANDS is the TEMPLATE STRAND. - TEMPLATE (ANTISENSE) strand is TRANSCRIBED INTO mRNA.
- CODING (SENSE) strand – nt BASE SEQUENCE CORRESPONDS
to RNA made, is the reference strand, 5’ – 3’ - Coding region often contains: EXONS interrupted by
INTRONS, intervening sequences between exons. - RNA POLYMERASE is the ENZYME THAT TRANSCRIBES THE RNA, USING COMPLEMENTARY BASE PAIRING WITH THE TEMPLATE STRAND TO GENERATE THE CORRECT RNA.
