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.

Figure 7-30 Examples of peptide sequence alignments.P

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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.

Figure 7-30 Examples of peptide sequence alignments.

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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.

Figure 7-38 A DNA chip.

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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.

Figure 7-40 Variation in the expression of genes that encode proteins known as cyclins (Section 34-4C) in human

tissues.

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Nucleic Acid Structure (Ch 27)• Double helical DNA has 3 major helical forms• B-DNA• A-DNA• Z-DNA

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Figure 29-1a

Structure 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.

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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.

Figure 29-2aStructure 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.

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A-DNA• 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.

Figure 29-3a

Structure 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.

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Z-DNA• 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.

Figure 29-5 X-Ray structure of two ADAR1 Z domains in complex with Z-DNA.

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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• 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 A-DNA (11 Å).

– Ribose rings have conformations similar to both A-DNA and B-DNA.

Figure 29-6 X-Ray structure of a 10-bp RNA–DNA hybrid helix consisting of d(GGCGCCCGAA) in complex

with r(UUCGGGCGCC).

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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.

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Glycosidic bond

Angles of sugar-phosphate 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.

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.

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