Genes

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Genes

• Introduction to Bioinformatics
• BM131/BM511
• Gary J. Schoenhals
• BMB
• University of Southern Denmark

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Definition of a gene
a segment of DNA found on a chromosome that codes
for a particular protein a unit of heredity
genes were formerly called factors
Source HyperDictionary (http//www.hyperdictionar
y.com)
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Structure of DNA
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Sugar backbone - Deoxyribose
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Purines
Base pairing
Base pairing
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Pyrimidines
Base pairing
Base pairing
in RNA
in DNA
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Base-pairing overview
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Base-pairing of nucleotides
3 hydrogen bond pairs
2 hydrogen bond pairs
Oxygen (red) Nitrogen (blue) Carbon (gray)
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DNA vs. RNAstructure
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Sugar backbone - Ribose
DNA
RNA
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Uracil (RNA) vs. Thymine (DNA)
in RNA Uracil
in DNA Thymine
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Second position
U
C
A
G
U
STOP
STOP
STOP
C
Third position
First position
A
G
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Genetic code - observations

• Redundancy most amino acids are encoded by more
  than one codon
• Mutations in the third position are often
  silent (result in the same amino acid)
• Mutations in the first or second position, though
  not usually silent, often result in substitution
  of an amino acid with similar characteristics
• The genetic code is robust and resistant to
  lethal changes

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Mutations
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tRNA
Amino acid binding
mRNA/ribosome interaction
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Prokaryotic
Eukaryotic
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Gene expression overview
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Prokaryote vs. Eukaryote
Note Localization of specific molecules can be
extremely important, especially in predictive
bioinformatics (e.g. GO database)
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Eukaryotes
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Eukaryotic Gene Structure
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Gene Regulation in Eukaryotes

• Altering rate of transcription
• Altering RNA processing while still in nucleus
• Altering stability of mRNA molecules (degradation
  rate) RNA interference
• Altering translation of mRNA by ribosomes
• Riboswitches some metabolites bind mRNA,
  affecting translation of the transcript, or cause
  enhanced transcription termination

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RNA Processing

• Transcription forms pre-mRNA
• Capping with modified guanine at 5 end
• protects against degradation
• Intron removal
• RNA editing in some organisms
• Synthesis of poly-A tail (stretch of adenine
  residues)
• Export to cytoplasm

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Introns/ExonsOverview
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RNA Processing
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Alternative splicing of RNA
Different proteins from the same gene!
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Characteristics

• Chicken collagen 52 exons
• Human dystrophin 79 exons
• Average exon is 140 nucleotides long, but introns
  can be quite large (e.g. 480 kbp!)
• Splicing done with spliceosome
• snRNA (small nuclear RNA) molecules plus approx.
  145 proteins
• approx. 12 different snRNA
• Disorders retinitis pigmentosa, spinal muscular
  atrophy
• http//www.neuro.wustl.edu/neuromuscular/pathol/sp
  liceosome.htm

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Spliceosome
U1 binds to 5' splice site
U2 binds to branchpointrecognition sequence
U4-U5-U6 complexbinds to 5' splice site
U5 binds exon sequencesIntron is removed
U1 is displacedU6 binds to U25'-splice site
near branchpoint
http//www.neuro.wustl.edu/neuromuscular/pathol/sp
liceosome.htm
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Spliceosome contd.

• Exonic splicing enhancers (ESEs) and exonic
  splicing silencers (ESSs) are contained in exons
  - these help regulate how splicing is done (e.g.
  specificity)
• Intronic splicing enhancers (ISEs) and intronic
  splicing silencers (ISSs) are contained in
  introns and function in a similar fashion to
  their exonic counterparts

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Enhancers
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Transcription factors modulate gene expression
Silencers are control regions of DNA which may
be far away from the gene, but when transcription
factors bind to them gene expression is repressed.
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Insulators
T-cell receptor for antigen gamma/delta encoding
region
Stretch of DNA that separates genes from one
another shielding them from the effects of
activation or repression of neighboring genes
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Prokaryotes
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Prokaryotic Genes are Arranged in Operons

• Genes arranged in operons
• Polycistronic mRNA
• One promoter but separate ribosome binding sites
• Used in predictive bioinformatics

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Lac Operon Control
cAMP low
cAMP high
cAMP high
cAMP low
CAP catabolite activator protein (binds
cAMP) cAMP cyclic adenosine monophosphate lac
repressor inactivated when bound to lactose
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CAP is a dimer that binds DNA and is the size of
one turn of the helix
Structural bioinformatics
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Inverted repeats important for CAP binding
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Transcription Factor Domains

• Only a few major types
• Bind DNA
• 3D conformation of binding domain recognizes DNA
  structure
• Interact with RNA polymerase
• Modulate transcription of genes understanding
  how to control a cells activities is an
  important part of drug discovery and development

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DNA Recognition Domains

• Helix-turn-helix motif
• Zinc finger motif
• Leucine zipper motif

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Restriction Enzymes

• Restriction enzymes recognize specific DNA
  sequences
• Bind to DNA
• Introduce a cut can be used for cloning
• E.g. BamHI (GGATCC) and HincII (GTCGAC)

?
?
5G GATCC3 3CCTAG G5
5GTC GAC3 3CAG CTG5
overhang generated sticky end
no overhang blunt end
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DNA Methylases

• Enzymes that modify DNA via methylation of bases
• Protects DNA from nucleases
• If methylation occurs at a restriction enzyme
  site, cutting could be inhibited
• E.g. TaqI methylase methylates TCGA

CH3
5GTCGAC3 3CAGCTG5
Enzyme HincII inhibited (GTCGAC)
CH3
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Bioinformatics Strategies

• Sequence alignment of DNA or proteins
• Used to find homologs
• Orthologs vs. paralogs
• Similarity/identity can imply conserved function
• Depending on the context, it may be better to use
  protein sequence rather than DNA
• Codon usage
• Gene prediction
• Motif searches
• Consensus sequences
• Secondary structure e.g. hairpin loops
• Presence of protein domains imparting
  functionality
• Phylogenetic analysis

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Alignments - Protein vs. DNA
Consider the two following DNA sequences
ATG CTT CCC TTG CAT TTT AAA Seq 1 ATG CTG CCG
CTC CAC TTC AAG Seq 2
Translation yields the following protein
sequences
Met Leu Pro Leu His Phe Lys Seq 1translated Met
Leu Pro Leu His Phe Lys Seq 2translated
Both DNAs encode identical protein sequences, but
Seq 1 shares only 14/21 bases with Seq 2 66.7
identity
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Codon Usage

• Use of certain codons to encode amino acids is
  non-random
• Highly expressed genes use a restricted set of
  codons for optimal translational efficiency
• Can be used to predict highly expressed genes
• Atypical codon usage implies horizontal gene
  transfer
• CodonW software can calculate Codon Adaptation
  Index (CAI), Codon Bias Index (CBI), etc.
• Some tools here
• http//bioweb.pasteur.fr/seqanal/dna/intro-uk.html

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Gene Prediction

• Prediction of open reading frames (ORFs) which
  represent the possibly expressed genes
• Can then obtain a list of theoretical proteins
  encoded by the genome via translation
• Some examples of tools for gene prediction
  include GlimmerHMM (eukaryotic genes) and Glimmer
  (prokaryotic genes)
• See The Institute for Genomic Research (TIGR) on
  the web at http//www.tigr.org/

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Motif Searches

• Searching for patterns with biological
  significance
• Examples include promoter sequences, enhancers,
  terminators
• Hidden Markov models (HMMs) are quite often
  employed in these types of searches
• Software examples ELPH (motifs), RBSfinder
  (ribosome binding sites)
• Prosite search engine for protein families and
  domains

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E. coli Promoter Consensus Sequences
s Factor Promoter Consensus Sequence
-35 Region
-10 Region s70 TTGACA
TATAAT s32 TCTCNCCCTTGAA CCCCATNTA
s28 CTAAA CCGATAT
-24 Region -12
Region s54 CTGGNA TTGCA
-10 region is also called Pribnow box, after its
discoverer
N any (A, T, C, or G)
E. coli has 5 different sigma factors, including
s38
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Transcription Factor Consensus Sequences
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Phylogenetic Analysis

• Use of conserved sequences to aid in
  classification of organisms
• Must choose sequences encoding molecules that
  have conserved function across species
• Evolutionary chronometer concept
• The difference between two sequences can be
  proportional to the evolutionary distance between
  those organisms
• Prokaryotes 16S rRNA, eukaryotes 18S rRNA

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Nucleotide Databases
DNA DataBank of Japan (DDBJ) Ensembl joint
between EBI and Wellcome Trust Sanger
Institute European Bioinformatics Institute
(EBI) European Molecular Biology Laboratory
(EMBL) Japan Biological Information Research
Center (JBIRC) National Center for Biotechnology
Information (NCBI) Additional Proteomics sites
or databases ExPASy Swiss-Prot Many others!
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Cross-reference
Protein sequence
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DNA sequence
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Database problems

• Incomplete annotation
• Missing information such as function, keywords,
  etc.
• Consequence a given search will likely not
  return all relevant database entries
• Redundancy
• Smaller DNA segments often included in larger
  ones (such as chromosome)
• ESTs (Expressed sequence tags)
• A database is only as good as the person(s)
  maintaining it garbage in garbage out!

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Scroll to bottom to see what is on next slide.
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Bit score (BLAST)
Colored bars represent matching portion of gene
color indicates functional group
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BLAST results