Introduction to Molecular Biology and Genomics

1
Introduction to Molecular Biology and Genomics

• Part One of a Short Course Series
• Functional Genomics and Computational Biology
• Lecture 2, October 26

2
Sources of Info

• Reading DNA Science, A First Course in
  Recombinant DNA Technology, Micklos and Freyer,
  CSH Press 1990
• Graphics http//www.accessexcellence.org
• DNA interactive model http//www.umass.edu/microb
  io/chime/dna/index.htm
• Glossary of terminology http//www.nhgri.nih.gov/
  DIR/VIP/Glossary/index.html

3
Summary of DNA structure features

• Double stranded helix, sugar-phosphate backbone
• Hydrogen bonding between bases maintains
  structure
• A-T and G-C only, but any order
• colinearity and self replication information
• Polarity of polymer 5 end and 3 end

4
Go to Netscape and Chime
5
Summary of DNA structure features

• Double stranded helix, sugar-phosphate backbone
• Hydrogen bonding between bases maintains
  structure
• A-T and G-C only, but any order
• colinearity and self replication information
• Polarity of polymer 5 end and 3 end

6
Biological Information Flow Central Dogma
TACTGACGAAAA ATGACTGCTTTT
EVERY CELL THE SAME
DNA
transcription
splicing (higher organisms)
RNA
AUGACUGCUUUU
DIFFERENT FUNCTION DIFFERENT EXPRESSION
translation
Protein
Met-Thr-Ala-Phe
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OUTLINE

• Macromolecular Synthesis
• Replication DNA to DNA
• Transcription DNA to RNA
• RNA processing
• Translation RNA to Protein
• Regulation of Transcription and Translation
• Cis-acting elements Promoters, operators,
  enhancers
• Trans-acting elements transcription factors
• Post translational Regulation and Signal
  Transduction

8
Cell Cycle and Replication

• Must decide if cycling (dividing) or not (G0/G1)
• Must copy genome once and only one before
  dividing (G1/S and S)
• Must result in one copy per daughter cell (M,
  mitosis)

G0
M
G2
G1
S
Eukaryotic Cell Cycle
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DNA Replication

• Replication is semiconservative
• Meselson and Stahl 1958

Next Generations assuming semiconservative
15N DNA (heavy)
Mix AC
Add 14N
Density Gradient
C
D
A
B
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DNA Replication

• Replication is semiconservative
• Meselson and Stahl 1958
• Polymerization is template dependent, uses DNA
  polymerases and requires nucleotide triphosphates
  as monomers
• Kornberg 1958
• Polymerization is primer dependent and
  directional 5-3
• Starts at origins of replication in vivo
• species-specific accessory proteins ID origin and
  create a bubble in the helix to get things started

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DNA Replication

• Replication is semiconservative
• Meselson and Stahl 1958
• Polymerization is template dependent, uses DNA
  polymerases and requires nucleotide triphosphates
  as monomers (dNTPs dGTP, dATP, dCTP, dTTP)
• Kornberg 1958
• Polymerization is primer dependent and
  directional 5-3

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Directionality and Primer Constraints
5
3
Double stranded helix uncoiled locally and
stabilized by accessory proteins single strand
binding protein and helicase. Tangles get
resolved by topoisomerases
Fork traveling in this direction
5
3
13
Directionality and Primer Constraints
5
3
DNA polymerases require a 3-OH and basepairing
at the end position Primase can join 2 NTPs de
novo, uses single stranded region as template to
generate small lengths of new RNA to act as
primers for DNA polymerase
Fork traveling in this direction
5
3
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Directionality and Primer Constraints
5
3
Asymmetrical fork New DNA in red leading
strand synthesis proceeds faster then lagging
strand because RNA primers must be removed
(RNAse H) and the gaps must be repaired, and
finally the fragments joined together (DNA
ligase)
Fork traveling in this direction
5
3
15
DNA Replication

• Replication is semiconservative
• Meselson and Stahl 1958
• Polymerization is template dependent, uses DNA
  polymerases and requires nucleotide triphosphates
  as monomers
• Kornberg 1958
• Polymerization is primer dependent and
  directional 5-3
• Starts at origins of replication in vivo
• species-specific accessory proteins ID origin and
  create a bubble in the helix to get things started

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(No Transcript)
17
TranscriptionMaking RNA from DNA
18
(No Transcript)
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Transcription Extracting Information from Storage

• DNA is relatively static storage media
• Transcription selectively converts information to
  useable format
• Uses are varied but mainly for structural
  components or for protein synthesis
• Genomes are organized as transcriptional units
• RNA is unstable in RNAse world so dynamic
  populations are the norm

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Transcription

• Bacterial
• coupled to translation
• Single RNA can encode more than one protein
• Eucaryotic
• Primary transcript
• Splicing
• caps
• polyadenylation

21
RNA Polymerase (RNAP)

• Viruses Usually encode their own RNAP
• Bacteria Use a single RNAP for all types of RNA
  synthesis
• Eukaryotes
• pol I transcribes small RNAs including tRNAs
  and snRNAs
• pol II transcribes protein encoding RNAs
• pol III transcribes rRNAs
• Each has unique signals in the DNA to recruit the
  correct enzyme

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Transcription
23
Types of RNA molecules

• ribosomal RNA (rRNA)
• transfer RNA (tRNA)
• small nuclear RNA (snRNA)
• heteronuclear RNA (hnRNA)
• messenger RNA (mRNA)

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rRNAs

• Structural components of ribosomes, part of the
  protein synthesis machinery
• 5S, 16S and 23S in bacteria, 5S, 5.8S, 18S and
  28S in eukaryotes (S measure of sedimentation
  rate relative to 3D shape)
• Encoded by multiple clustered genes as a
  precurser transcript that gets processed
• Transcription visible as dense regions of
  nucleolus, a subregion of the cell nucleus

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tRNA

• Involved in translation as adapter molecule
• Family of RNAs with similar structure
• Some very conserved regions
• transcribed in nucleolus by pol I
• Highly modified post-transcriptionally to
  generate additional nucleotide types

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Messenger RNA

• Procaryotes
• Encodes instructions for protein synthesis
• physically coupled to protein synthesis machinery
  in procaryotes, immediately decoded
• Eukaryotes
• Primary transcript (hnRNA) is exact replica of
  coding strand of DNA (TgtU substitution)
• coding sequences (exons) interlaced with
  noncoding sequences (introns) which are removed
  by splicing
• Additional processing to generate a mature
  message by addition of 5 7meG cap and
  polyadenylate tail

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Higher Resolution mRNA Processing
intron1
exon 3
exon 1
exon 2
intron 2
DNA
Prot
ein
intron1
exon 1
exon 2
exon 3
intron 2
RNA
7meG
Prot
ein
7meG
Prot
ein
AAAA(n)

7meG
AAAA(n)
Protein
Spliced mRNA
AAAA(n)
7meG
Protein
Mature mRNA
CAP
5UTR
CODING REGION
3UTR
polyA tail
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Transcription Extracting Information from Storage

• DNA is relatively static storage media
• Transcription selectively converts information to
  useable format. This is the major source of
  functional diversity
• Uses are varied but mainly for structural
  components or for protein synthesis
• Genomes are organized as transcriptional units
• RNA is unstable in RNAse world so highly
  regulated dynamic populations are the norm

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TranslationDecoding RNA to make Proteins
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Protein SynthesisTranslation

• Parts List
• mRNA is template
• tRNA
• ribosomes
• amino acids
• aminoacyl tRNA transferases

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Protein polypeptide

• Polymer of amino acids
• 20 letter alphabet
• sequence of amino acids is colinear with DNA
  sequence
• amno acids attached by peptide bond

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Amino Acids monomers of protein
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Amino Acids monomers of protein
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Amino Acids monomers of protein
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Amino Acids monomers of protein
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Amino Acid 3-letters 1-letter Alanine
Ala
A Cysteine
Cys C Aspartic
Acid Asp
D Glutamic Acid Glu
E Phenylalanine
Phe
F Glycine Gly
G Histidine
His
H Isoleucine Ile
I Lysine
Lys
K Leucine Leu
L Methionine
Met
M Asparagine Asn
N Proline
Pro
P Glutamine Gln
Q Arginine
Arg
R Serine Ser
S Threonine
Thr
T Valine Val
V Tryptophan
Trp W Tyrosine
Tyr Y
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Peptide Bond also directional
Peptide bond
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Protein SynthesisTranslation

• Parts List
• mRNA is template
• tRNA
• ribosomes
• amino acids
• aminoacyl tRNA transferases

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Reminder of mRNA structure
CAP
5UTR
CODING REGION
3UTR
polyA tail
40
tRNA

• Involved in translation as adapter molecule
  (Crick predicted this and experimentally
  suggested they recognized a triplet)
• Family of RNAs with similar structure
• Some very conserved regions
• transcribed in nucleolus by pol I
• Highly modified post-transcriptionally to
  generate additional nucleotide types
• Many different tRNAs sequenced leading to ID of
  anticodon which matched DNA

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tRNA amino acid carrier AND code reader
Each tRNA can specifically recruit an aminoacyl
tRNA synthetase to become charged with
the appropriate amino acid at the acceptor
sequence at 3 end Anticodon sequence in the
anticodon loop base pairs with the mRNA to
position amino acid adjacent to growing peptide
chain
42
tRNA is Adapter between mRNA and Protein Synthesis
43
Deciphering the Code

• Nirenburg (NIH) and Khorana (Wisconsin)
  independently developed cell free translation
  systems which could convert amino acids to
  polypeptides
• Nirenburg synthesized poly-uracil RNA and added
  to cell free system. This produced long chains of
  phenylalanine.
• Repeated with all combinations of sequences to
  determine code empirically

44
Decoding Codons to Amino Acids
45
Open Reading Frame ORF

• Due to code being a triplet
• there are three reading frames on each strand
  of DNA
• designated 1, 2, 3 and -1, -2 , -3 respectively
• Open reading frame is what is in between a start
• codon and a stop codon, usually only one of
• the six reading frames for any stretch of
  eukaryotic
• genome

CAP
Open Reading Frame (ORF)
5UTR
3UTR
polyA tail
Stop Codon
Start Codon
46
Ribosomes

• Two subunits 50S and 30S in procaryotes, 60S and
  40S in eukaryotes
• Mass is 60 RNA with 20-50 protein/subunit
• Many accessory factors for initiation,
  elongation, termination
• Small subunit engages mRNA then recruits large
  subunit
• Assembled ribosome/mRNA creates binding sites for
  aminoacyl-tRNAs P site and A site

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Mechanics of Translation



P
A
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Translation Summary

• mRNA is template
• Codons and anticodons of tRNAs
• Start Codons and Stop codons define protein
  coding region
• Codons define Reading Frame
• Ribosomes are the enzyme
• Truckloads of accessory proteins involved with
  initiation, elongation, and termination

49
Resources

• Transcription
• Genes VII, Lewin, Oxford University Press
• Translation
• Translation in Eukaryotes, Trachsel, CRC Press

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Regulation of Transcription and Post-Transcription
51
Regulation of Gene Expression
52
Transcriptional Regulation

• mRNA steady-state pool is function of rates of
  synthesis, processing, transport, and degradation
• Once initiated, elongation is rapid so initiation
  is major control point
• Initiation control elements consist of cis-acting
  and trans-acting elements
• Interrelated elements and genes give rise to
  cascades of gene regulation

53
Cis-acting Elements

• Cis equals attached elements in the DNA
• Promoter sum of all the cis acting elements
  for a operon (procaryotes) or gene (euk.)
• Promoter consists for core elements which are
  required for transcription and regulatory
  elements which alter initiation rates
• Core elements are highly conserved,
  polymerase-specific and wide spread
• Regulatory elements provide specificity of
  expression
• ALL are binding sites for proteins trans-acting

54
Cis elements

• Procaryotes operators, act by recruiting
  proteins () to the proximity of the RNA
  polymerase at the core promoters often contain
  binding sites for negative regulators called
  repressors
• Eukaryotes enhancers bind factors to increase
  initiation. Enhancers work at a distance, in any
  orientation (double stranded) and upstream or
  downstream initiation site. Recruiting protein
  which interact with others at the core promoter

55
Promoter Bashing

• Used to define cis-elements
• replace normal gene with reporter gene
• Assay different pieces of DNA

B-cell
core
ORF
A-cell
B-cell
core
reporter
A-cell
A B X
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Ectopic Regulation

• Cis elements can be moved to a novel location in
  the genome and change the expression of the
  downstream gene
• Cis elements have independent and additive
  effects weak core promoters can be replaced
  with strong core promoters to increase
  expression but specificity is maintained by
  regulatory elements

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Example Gene Therapy

• Need to replace a defective enzyme only in cells
  of the immune system
• Identify immune cell-specific gene expression
• Identify the regulatory elements responsible
• Place elements upstream of strong promoter and
  correct enzyme gene and reintroduce into immune
  cells

58
Trans-acting Transcription Factors

• Multidomain functions can be assigned to regions
  of the protein. Domains can often be swapped
  around
• Activities include proteinDNA interactions and
  proteinprotein interactions
• DNA binding helix-turn-helix,
  helix-loop-helix, zinc-finger
• Protein binding leucine zipper

59
Domain swapping

• Create chimeric proteins protein fusion to
  demonstrate specificity
• Activation domains have broad activity due to
  common target of pol II

Act
Act
DNA
DNA
Act
Pol II
DNA
OK
Reporter 1
Act
Pol II
DNA
Reporter 2
OK
60
Transcriptional Regulation

• mRNA steady-state pool is function of rates of
  synthesis, processing, transport, and degradation
• Once initiated, elongation is rapid so initiation
  is major control point
• Initiation control elements consist of cis-acting
  and trans-acting elements
• Interrelated elements and genes give rise to
  cascades of gene regulation

61
Cascade or Program of Gene expression
Enzymes/structural proteins
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Post-Transcriptional Regulation
63
Post-Transcriptional Regulation

• Examples of regulation at every step demonstrated
• RNA stability 5 UTR and 3 UTR can contain
  sequences giving rise to protein binding
  structures which increase or decrease half life
  of the RNA
• Translational control
• RNA storage maternal messages, membrane
  biosynthesis
• Attenuation mechanisms alterative structures
  dependent on rate of translation

64
Post-Transcriptional Regulation
65
Post-Transcriptional Regulation