FCH 532 Lecture 9

Chapter 7 Chapter 29 Exam Friday!

 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.

BLAST

Figure 7-30

Examples of peptide sequence alignments.

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 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.

FASTA

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.

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.

Figure 7-39

The photolithographic synthesis of a DNA chip.

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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 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.

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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 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 29-2a 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.

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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 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.

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 . 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 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 Base pairs inclined to the helical axis by 13.9 B-DNA like qualities:
Minor groove is intermediate (9.5 ) between B-DNA (7.4 ) and ADNA (11 ). Ribose rings have conformations similar to both A-DNA and B-DNA.

Figure 29-6 X-Ray structure of a 10-bp RNADNA 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.

Glycosidic bond
Angles of sugarphosphate backbone
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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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