Microbiology 7/e

1
Chromosomes

• Chromosomes
• Strands of DNA that contain all of the genes an
  organism needs to survive and reproduce

• Genes
• Segments of DNA that specify how to build a
  protein
• genes may specify more than one protein in
  eukaryotes
• Chromosome maps are used to show the locus
  (location) of genes on a chromosome

2
Chromosomes

• Genetic Variation
• Phenotypic variation among organisms is due to
  genotypic variation (differences in the sequence
  of their DNA bases)
• Differences exist between species and within a
  species
• Different genes (genomes) ? different proteins
  (proteomes)
• Different versions of the same gene (alleles)
• Differences in gene expression (epigenetics)

3
DNA Replication

• Cell Division (mitosis)
• Cells must copy their chromosomes (DNA synthesis)
  before they divide so that each daughter cell
  will have a copy
• A region of the chromosome remains uncopied
  (centromere) in order to hold the sister
  chromatids together
• Keeps chromatids organized to help make sure each
  daughter cell gets exactly one copy
• Nondisjunction is when sister chromatids do not
  assort correctly and one cell ends up with both
  copies while the other cell ends up with none

4
DNA Replication

• DNA Synthesis
• The DNA bases on each strand act as a template to
  synthesize a complementary strand
• Recall that Adenine (A) pairs with thymine
  (T)and guanine (G) pairs with cytosine (C)
• The process is semiconservative because each new
  double-stranded DNA contains one old strand
  (template) and one newly-synthesized
  complementary strand

5
DNA Replication

• DNA Polymerase
• Enzyme that catalyzes the covalent bond between
  the phosphate of one nucleotide and the
  deoxyribose (sugar) of the next nucleotide

DNA Polymerization
6
DNA Replication

• 3 end has a free deoxyribose
• 5 end has a free phosphate
• DNA polymerase
• can only build the new strand in the 5 to 3
  direction
• Thus scans the template strand in 3 to 5
  direction

7
DNA Replication

• Initiation
• Primase (a type of RNA polymerase) builds an RNA
  primer (5-10 ribonucleotides long)
• DNA polymerase attaches onto the 3 end of the
  RNA primer

8
DNA Replication

• Elongation
• DNA polymerase uses each strand as a template in
  the 3 to 5 direction to build a complementary
  strand in the 5 to 3 direction

DNA polymerase
9
DNA Replication

• Elongation
• DNA polymerase uses each strand as a template in
  the 3 to 5 direction to build a complementary
  strand in the 5 to 3 direction
• results in a leading strand and a lagging strand

10
DNA Replication

• Leading Strand
• Topisomerase unwinds DNA and then Helicase breaks
  H-bonds
• DNA primase creates a single RNA primer to start
  the replication
• DNA polymerase slides along the leading strand in
  the 3 to 5 direction synthesizing the matching
  strand in the 5 to 3 direction
• The RNA primer is degraded by RNase H and
  replaced with DNA nucleotides by DNA polymerase,
  and then DNA ligase connects the fragment at the
  start of the new strand to the end of the new
  strand (in circular chromosomes)

11
DNA Replication

• Lagging Strand
• Topisomerase unwinds DNA and then Helicase breaks
  H-bonds
• DNA primase creates RNA primers in spaced
  intervals
• DNA polymerase slides along the leading strand in
  the 3 to 5 direction synthesizing the matching
  Okazaki fragments in the 5 to 3 direction
• The RNA primers are degraded by RNase H and
  replaced with DNA nucleotides by DNA polymerase
• DNA ligase connects the Okazaki fragments to one
  another (covalently bonds the phosphate in one
  nucleotide to the deoxyribose of the adjacent
  nucleotide)

12
DNA Replication
Topoisomerase - unwinds DNA Helicase enzyme
that breaks H-bonds DNA Polymerase enzyme that
catalyzes connection of nucleotides to form
complementary DNA strand in 5 to 3 direction
(reads template in 3 to 5 direction) Leading
Strand transcribed continuously in 5 to 3
direction Lagging Strand transcribed in
segments in 5 to 3 direction (Okazaki
fragments) DNA Primase enzyme that catalyzes
formation of RNA starting segment (RNA
primer) DNA Ligase enzyme that catalyzes
connection of two Okazaki fragments
13
Web Resources

• DNA Replication (synthesis)
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter11/animation_quiz_2.html
• http//www.wiley.com/college/pratt/0471393878/stud
  ent/animations/dna_replication/index.html
• http//www.biostudio.com/d_20DNA20Replication20
  Coordination20Leading20Lagging20Strand20Synthe
  sis.htm
• http//www.biostudio.com/d_20DNA20Replication20
  Nucleotide20Polymerization.htm
• http//www.dnalc.org/resources/3d/DNAReplicationBa
  sic_w_FX.html (download this video file from
  the website to view it without interruptions)
• http//www.stolaf.edu/people/giannini/flashanimat/
  molgenetics/dna-rna2.swf
• http//www.bioteach.ubc.ca/TeachingResources/Molec
  ularBiology/DNAReplication.swf

14
Protein Synthesis

• DNA provides the instructions for how to build
  proteins
• Each gene dictates how to build a single protein
  in prokaryotes
• The sequence of nucleotides (AGCT) in DNA dictate
  the order of amino acids that make up a protein

15
Protein Synthesis

• DNA provides the instructions for how to build
  proteins
• Each gene dictates how to build a single protein
  in prokaryotes
• The sequence of nucleotides (AGCT) in DNA dictate
  the order of amino acids that make up a protein

16
Protein Synthesis

• Protein synthesis occurs in two primary steps

17
Protein Synthesis

• Transcription Initiation
• RNA polymerase binds to a region on DNA known as
  the promoter, which signals the start of a gene
• Promoters are specific to genes
• RNA polymerase does not need a primer
• Transcription factors assemble at the promoter
  forming a transcription initiation complex
  activator proteins help stabilize the complex
  
• Gene expression can be regulated (turned on/off
  or up/down) by controlling the amount of each
  transcription factor

18
Protein Synthesis

• Transcription Elongation
• RNA polymerase unwinds the DNA and breaks the
  H-bonds between the bases of the two strands,
  separating them from one another
• Base pairing occurs between incoming RNA
  nucleotides and the DNA nucleotides of the gene
  (template)
• recall RNA uses uracil instead of thymine

AGTCAT
UCA
GUA
19
Protein Synthesis

• Transcription Elongation
• RNA polymerase unwinds the DNA and breaks the
  H-bonds between the bases of the two strands,
  separating them from one another.
• Base pairing occurs between incoming RNA
  nucleotides and the DNA nucleotides of the gene
  (template)
• recall RNA uses uracil instead of thymine
• RNA polymerase catalyzes bond to form between
  ribose of 3 nucleotide of mRNA and phosphate of
  incoming RNA nucleotide

5
3
ATP
5
3
ADP
20
Protein Synthesis

• Transcription Elongation

The gene occurs on only one of the DNA strands
each strand possesses a separate set of genes
21
Protein Synthesis

• Transcription Termination
• A region on DNA known as the terminator signals
  the stop of a gene
• RNA polymerase disengages the mRNA and the DNA

22
Protein Synthesis

• Exons are coding regions
• Introns are removed
• different combinations of exons form different
  mRNA resulting in multiple proteins from the same
  gene
• Humans have 30,000 genes but are capable of
  producing 100,000 proteins

• Alternative Splicing (eukaryotes only)

23
Web Resources

• Transcription
• http//www.biostudio.com/d_20Transcription.htm
• http//www.youtube.com/watch?vWsofH466lqk
• http//www.dnalc.org/resources/3d/TranscriptionBas
  ic_withFX.html

• Alternative Splicing
• http//www.youtube.com/watch?vFVuAwBGw_pQfeature
  related

24
Protein Synthesis
2
mRNA

• mRNA is used by ribosome to build protein
• (Ribosomes attach to the mRNA and use its
  sequence of nucleotides to determine the order of
  amino acids in the protein)
• Cytoplasm of prokaryotes and eukaryotes
• Some proteins feed directly into rough ER in
  eukaryotes

25
Protein Synthesis

• Translation
• Every three mRNA nucleotides (codon) specify an
  amino acid

26
Protein Synthesis

• Translation
• tRNA have an anticodon region that specifically
  binds to its codon

27
Protein Synthesis

• Translation
• Each tRNA carries a specific amino acid

28
Protein Synthesis
Aminoacyl tRNA synthetases attach amino acids to
their specific tRNA
29
Protein Synthesis

• TranslationInitiation
• Start codon signals where the gene begins (at 5
  end of mRNA)

5
3
AUGGACAUUGAACCG
start codon
30
Protein Synthesis
Small ribosomal subunit

• TranslationInitiation
• Start codon signals where the gene begins (at 5
  end of mRNA)
• Ribosome binding site (Shine Dalgarno sequence)
  upstream from the start codon binds to small
  ribosomal subunit
• then this complex recruits the large ribosomal
  subunit

Small ribosomal subunit
31
Protein Synthesis

• TranslationScanning
• The ribosome moves in 5 to 3 direction
  reading the mRNA and assembling amino acids
  into the correct protein

large ribosome subunit
small ribosome subunit
32
Protein Synthesis

• TranslationScanning
• The ribosome moves in 5 to 3 direction
  reading the mRNA and assembling amino acids
  into the correct protein

33
Protein Synthesis

• TranslationTermination
• Ribosome disengages from the mRNA when it
  encounters a stop codon

34
Web Resources

• Translation
• Eukaryotic http//www.youtube.com/watch?v5bLEDd
  -PSTQfeaturerelated
• Prokaryotic http//www.biostudio.com/d_20Protei
  n20Synthesis20Prokaryotic.htm
• http//www.biostudio.com/d_20Peptide20Bond20For
  mation.htm
• http//www.johnkyrk.com/DNAtranslation.html
• http//www.dnalc.org/resources/3d/TranslationBasic
  _withFX0.html
• http//www.dnalc.org/resources/3d/TranslationAdvan
  ced.html

35
Practice Question
Translate the following mRNA sequence AGCUACCAUACG
CACCCGAGUUCUUCAAGC
36
Practice Question
Translate the following mRNA sequence AGCUACCAUACG
CACCCGAGUUCUUCAAGC
Serine Tyrosine Histidine Threonine
Histidine Proline Serine Serine Serine -
Serine
37
Practice Question
Translate the following mRNA sequence AGCUACCAUACG
CACCCGAGUUCUUCAAGC
Serine Tyrosine Histidine Threonine
Histidine Proline Serine Serine Serine -
Serine
Ser Tyr His Thr His Pro Ser Ser
Ser - Ser
38
Practice Question
Translate the following mRNA sequence AGCUACCAUACG
CACCCGAGUUCUUCAAGC
Serine Tyrosine Histidine Threonine
Histidine Proline Serine Serine Serine -
Serine
Ser Tyr His Thr His Pro Ser Ser
Ser - Ser
S Y H T H P S S S - S
39
Protein Synthesis
Translation

• Multiple RNA polymerases can engage a gene at one
  time
• Multiple ribosomes can engage a single mRNA at
  one time

Transcription
40
Protein Synthesis

• Eukaryotes transcription occurs in the nucleus
  and translation occurs in the cytoplasm
• Prokaryotes Transcription and translation occur
  simultaneously in the cytoplasm

41
RNA

• There are four main types of RNA
• mRNA - RNA copy of a gene used as a template for
  protein synthesis
• rRNA - part of structure of ribosomes
• tRNA- amino acid carrier that matches to mRNA
  codon
• snRNA - found in nucleus where they have several
  important jobs

42
Practice Questions

1. Why is DNA synthesis said to be
   semiconservative?
2. What role do DNA polymerase, DNA primase (a type
   of RNA polymerase), helicase, topoisomerase,
   RNase H, and ligase play in DNA replication?
3. What is the difference between how the leading
   strand and lagging strand are copied during DNA
   replication? Why do they have to be synthesized
   differently in this fashion?
4. What would happen if insufficient RNase H were
   produced by a cell? What if insufficient ligase
   were produced by a cell?
5. What are four key differences between DNA
   polymerase and RNA polymerase? (they are
   difference molecules doesnt count as one!)
6. Compare and contrast codons and anticodons?
7. What is alternative splicing? Why is it necessary
   in eukaryotes?
8. During translation, what amino acid sequence
   would the following mRNA segment be converted
   into AUGGACAUUGAACCG?
9. How come there are only 20 amino acids when there
   are 64 different codons?
10. How come prokaryotes can both transcribe and
    translate a gene at the same time, but eukaryotes
    cannot?

43
Web Resources

• Transcription
• http//www.biostudio.com/d_20Transcription.htm
• http//www.youtube.com/watch?vWsofH466lqk
• http//www.dnalc.org/resources/3d/TranscriptionBas
  ic_withFX.html

• Alternative Splicing
• http//www.youtube.com/watch?vFVuAwBGw_pQfeature
  related

• Translation
• Eukaryotic http//www.youtube.com/watch?v5bLEDd
  -PSTQfeaturerelated
• Prokaryotic http//www.biostudio.com/d_20Protei
  n20Synthesis20Prokaryotic.htm
• http//www.biostudio.com/d_20Peptide20Bond20For
  mation.htm
• http//www.johnkyrk.com/DNAtranslation.html
• http//www.dnalc.org/resources/3d/TranslationBasic
  _withFX0.html
• http//www.dnalc.org/resources/3d/TranslationAdvan
  ced.html

44
Web Resources
Insulin Example of Protein Synthesis http//www.bi
otopics.co.uk/as/insulinproteinstructure.html
Hemoglobin Example of Protein Synthesis http//www
.biotopics.co.uk/as/insulinproteinstructure.html
Collagen Example of Protein Synthesis http//www.b
iotopics.co.uk/JmolApplet/collagen.html
45
Images

• http//www.kscience.co.uk/as/module1/pictures/bact
  eria.jpg
• http//www.biologie.uni-hamburg.de/b-online/librar
  y/onlinebio/14_1.jpg
• http//pharmamotion.com.ar/wp-content/uploads/2009
  /12/nrti_mechanism_action_antiretrovirals.jpg
• http//biology200.gsu.edu/houghton/4564202704/fi
  gures/lecture204/AAAreverse.jpg
• http//www.ebi.ac.uk/thornton-srv/databases/pdbsum
  /2d8x/traces.jpg
• http//www.ncbi.nlm.nih.gov
• http//xarquon.jcu.cz/edu/uvod/09nucleus/092functi
  on/images/activation3.jpg
• http//www.ncbi.nlm.nih.gov
• http//bass.bio.uci.edu/hudel/bs99a/lecture23/lec
  ture4_4.html
• http//selfhpvdna.diagcorlab.com/images/images/Cer
  vicalCancer.jpg