Transcript Lecture 27

FCH 532 Lecture 9
Chapter 7
Chapter 29
Exam Friday!
BLAST
• BLAST (basic local alignment search tool) and FASTA use
different search philosophies.
• BLAST (http://www.ncbi.nlm.nih.gob/BLAST/) performs pairwise
alignments up to user-selected number of subject sequences in the
selected database(s) most similar to the input query sequence.
• Can align vs ~900,000 peptide sequences in the database.
• Pairwise alignments are found using BLOSUM62 and listed
according to decreasing statistical significance.
• Alignments show both identical residues and similar residues
between the query sequence and aligned sequence and gaps will
be indicated.
• Assigns “E” value - expected value = number of expected results
by chance.
• The higher the E value, the less significant.
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Figure 7-30
Examples of peptide sequence alignments.
FASTA
• FASTA (http://www.ebi.ac.uk/fasta33/) allows users to
choose the substitution matrix (PAM, BLOSUM) the
default is BLOSUM50.
• Allows user to choose the gap penalty parameters.
• Allows user to choose ktup (k-tuple) value of 1 or 2 =
number of consecutive residues in “words” that FASTA
uses to search for identities.
• The smaller the ktup value, the more sensitive the
alignment.
CLUSTAL
• Multiple sequence alignment -To make alignments
with more than 2 sequences.
• CLUSTAL (http://www2.ebi.ac.uk/clustalw/)
• User can select matrix and gap penalties.
• Finds all possible pairwise alignments.
• Starting with the highest scoring pairwise alignment,
realigns remaining sequence.
• Should be looked at carefully.
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Figure 7-30
Examples of peptide sequence alignments.
Chemical synthesis of oligonucleotides
• Basic strategy is similar to polypeptide synthesis.
• Protected nucleotide is coupled to growing end of
oligonucleotide chain.
• Protecting group is removed.
• Process repeated until desired oligo has been synthesized.
• Current method is the phosphoramidite method
• Nonaqueous reaction sequence.
• 4 steps.
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1. Dimethoxytrityl (DMTr) protecting group at the 5’ end
is removed with trichloroacetic acid (Cl3CCOOH)
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2. The 5’ end of the oligo is couple to the 3’
phosphoramidite derivative. Tetrazole is used as
coupling agent.
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3. Any unreacted 5’ end group is capped by acetylation
to block its extension.
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4. The phosphite triester group
from the coupling step is
oxidized with I2 to the
phosphotriester.
Treated with NH4OH to remove
blocking groups.
DNA Chips
• Determination of the whole genomes from several organisms
allows us to ask significant questions about the function of all the
genes.
• Under what circumstances and to what extent is each gene
expressed under specific conditions?
• How do gene products interact to yield a functional organism?
• What are the consequences of variant genes?
• DNA chips (microarrays, gene chips) can be used for global
analysis of gene expression during biological responses.
• Arrays of different DNA oligonucleotides anchored to a glass or
nylon substrate in a grid.
• ~1 million oligonucleuotides can by simultaneously synthesized
using photolithography and DNA synthesis.
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Figure 7-38
A DNA chip.
DNA Chips
• Photolithography-oligonucleotides are synthesized with
photochemically removable protective groups at the 5’ end.
• Function in a similar manner as the DMTr group in conventional
synthesis.
• For the synthesis of a specific oligonucleotide, utilize masks that
protect specific oligos from being exposed to light while those that
are to be extended are exposed to light. (deprotection)
• The chip is then incubated with a solution of activated nucleotide
that couples only to the deprotected oligos.
• Excess is washed away and the process is repeated.
• Nanoliter sized droplets of reagents are applied using a device
similar to an ink jet printer.
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Figure 7-39
The photolithographic synthesis of a DNA
chip.
Applications: SNPs
• Can be used to examine single nucleotide
polymorphisms (SNPs)
• L-residue oligos are arranged in an array of L columns
by 4 rows for a total of 4L sequences.
• The probe in the Mth column has the standard
sequence with the exception of the probes Mth position
where it has a different base (A,C,G, or T) in each row.
• One probe is standard whereas the other three in each
column differ by one base pairs.
• The probe array is hybridized with complementary DNA
or RNA and variations in hybridization due to the SNPs
can be rapidly determined.
Applications: Expression profiles
• DNA features put onto a chip and the level of
expression of the corresponding genes in a tissue of
interest can be determined by the degree of
hybridization of its fluorescently labeled mRNA or cDNA
population.
• Used to generate an expression profile - pattern of
expression.
• Can be done with mRNA isolated under different growth
conditions.
• Can check how specific genes are affected.
• Example: cyclin gene expression in different tissues of
the same organism.
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Figure 7-40 Variation in the expression of genes that
encode proteins known as cyclins (Section 34-4C) in human
tissues.
Nucleic Acid Structure (Ch 27)
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Double helical DNA has 3 major helical forms
B-DNA
A-DNA
Z-DNA
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Figure 29-1a
Structur
e of B-DNA. (a)
Ball and stick
drawing and
corresponding
space-filling
model viewed
perpendicular to
the helix axis.
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Figure 29-1b Structure of B-DNA. (b) Ball and stick drawing and
corresponding space-filling model viewed down the helix axis.
B-DNA
• Dominant biological form.
• Right handed double helix
• Bases occupy the core, planes perpindicular to axis of
double helix
• Stacked via van der Waals contact.
• ~20 Å in diameter.
• Narrow minor groove.
• Wide major groove
• Ideal helical twist 10 bp/turn
• Watson-Crick base pairs in either orientation are
structurally interchangeable.
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Figure 29-2a
Structure
of A-DNA. (a) Ball
and stick drawing
and corresponding
space-filling model
viewed
perpendicular to
the helix axis.
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Figure 29-2b Structure of A-DNA. (b) Ball and stick drawing and
corresponding space-filling model viewed down the helix axis.
A-DNA
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Forms when relative humidity is reduced to 75% from B-DNA.
Reversible.
Wider and flatter right handed helix.
11.6 bp per turn, 34 Å pitch.
Planes of base pairs tilted 20° relative to the helical axis.
Deep major groove
Shallow minor groove
Only 2 biological examples:
– 3 bp segment present at the active site of DNA polymerase.
– Can be found in Gram-positive bacterial spores.
– B to A conformational changes inhibit UV cross-linking of pyrimidines.
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Figure 29-3a
Struct
ure of Z-DNA.
(a) Ball and
stick drawing
and
corresponding
space-filling
model viewed
perpendicular
to the helix
axis.
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Figure 29-3b Structure of Z-DNA. (b) Ball and stick drawing and
corresponding space-filling model viewed down the helix axis.
Z-DNA
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Observed by Wang and Rich d(CGCGCG)
Left-handed double helix
12 Watson-Crick base pairs per turn
Pitch 44 Å.
Deep minor groove
No major groove
Base pairs are flipped 180° relative to those of B-DNA
Repeating unit is a dinucleotide instead of a single nucleotide
Phosphate groups follow a zig-zag pattern.
Conditions: alternating purine/pyrimidine and high salt
Z-DNA binding protein (ADAR1) suggests that can also exist in
vivo.
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Figure 29-5 X-Ray structure of two ADAR1 Z
domains in complex with Z-DNA.
Duplex of self-complementary d(CGCGCG) hexamers
interacts with Z domains of ADAR1
RNA duplexes
• RNA is unable to make B-DNA because of steric hinderance from
the 2’-OH groups.
• Forms A-DNA-like structure called A-RNA or RNA-11
• 11 bp per turn
• Pitch 30.9 Å
• Base pairs inclined on helical axis by 16.7°
• Hybrid RNA-DNA duplexes are similar to both A-RNA and B-DNA
RNA-DNA hybrid duplexes
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Hybrid RNA-DNA duplexes are similar to both A-RNA and B-DNA
10.9 bp per turn
Pitch 31.3 Å
Base pairs inclined to the helical axis by 13.9°
B-DNA like qualities:
– Minor groove is intermediate (9.5 Å) between B-DNA (7.4 Å) and ADNA (11 Å).
– Ribose rings have conformations similar to both A-DNA and B-DNA.
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Figure 29-6 X-Ray structure of a 10-bp RNA–DNA
hybrid helix consisting of d(GGCGCCCGAA) in complex
with r(UUCGGGCGCC).
Sugar-phosphate chain conformations
• Double-stranded DNA has limited structural complexity compared
to proteins (only 4 nucleotides vs. 20 amino acids)
• Limited secondary structures, no tertiary or quaternary structures.
• RNA has some well-defined tertiary structure.
• Conformation of a nucleotide is specified by 6 torsion angles of
the sugar phosphate backbone and the torsion angle that
describes the orientation of the base about the glycosidic bond.(7
total).
• Despite 7 degrees of freedom per nucleotide, they have restricted
conformational freedom.
Figure 29-7 The conformation of a nucleotide unit is
determined by the seven indicated torsion angles.
Glycosidic bond
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Angles of
sugarphosphate
backbone
Torsion Angles about Glycosidic Bonds Have only 1 or 2
Stable Positions
• Purine residues have 2 sterically allowed orientations relative to
the ribose group, syn and anti
• For pyrimidines only the anti conformation is allowed due to
steric hinderance between the sugar and the C2 of the
pyrimidine.
• Most double hlical nucleic acids are in the anti conformation
• Exception is Z-DNA which has alternating anti and syn pyrimidine
and purine residues.
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Figure 29-8 The sterically allowed orientations of
purine and pyrimidine bases with respect to their
attached ribose units.
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Figure 29-1b Structure of B-DNA. (b) Ball and stick drawing and
corresponding space-filling model viewed down the helix axis.
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Figure 29-3b Structure of Z-DNA. (b) Ball and stick drawing and
corresponding space-filling model viewed down the helix axis.
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Figure 29-4 Conversion of
B-DNA to Z-DNA.