Microbial Genetics

1
Microbial Genetics
Chapter 9
2
Genetics the study of heredity

• transmission of biological traits from parent to
  offspring
• expression variation of those traits
• structure function of genetic material
• how this material changes

3
Levels of genetic study
4
What you need to know about microbial genetics

• Genetic structure/function of DNA, chromosomes,
  genes and genomes also including size and
  arrangement both prokaryotes and eukaryotes.
• Mechanisms of replication, transcription and
  translation including enzymes for proks., euks.
  and viruses.
• Gene regulation inducible vs. repressible
  operons (Prokaryotes only)
• Mutations types, causes and effects
• Recombination conjugation, transformation.
• Famous names in the history of genetics

5
Levels of structure function of the genome
Genetic structure/function

• ____________ sum total of genetic material of
  an organism (chromosomes mitochondria/chloroplas
  ts and/or plasmids)
• genome of cells DNA
• genome of viruses DNA or RNA
• ____________ length of DNA containing genes
• ____________ -fundamental unit of heredity
  responsible for a given trait
• site on the chromosome that provides information
  for a certain cell function (____________ )
• segment of DNA that contains the necessary code
  to make a protein or RNA molecule

6
Locations and forms of genomes
Genetic structure/function
7
Genomes vary in size
Genetic structure/function

• smallest virus 4-5 genes
• E. coli single chromosome containing 4,288
  genes 1 mm 1,000X longer than cell (4.5 Mbp)
• Human cell 46 chromosomes containing 31,000
  genes 6 feet 180,000X longer than cell

8
Genome packaging
Genetic structure/function

• Prokaryotes coiled into tight bundle by gyrase
  (a topoisomerase)
• Eukaryotes
• 1. wound around histone proteins to form
  nucleosomes
• 2. nucleosomes condense, coil into chromatin
  fibers
• 3. Chromatin supercoils and condenses into
  __________________

9
Genetic structure/function
10
Nucleic acid structure (RNA, DNA)
Genetic structure/function

• Nucleic acids are made of nucleotides (polymer,
  monomer)
• each nucleotide consists of 3 parts
• a 5 carbon _____________(deoxyribose or ribose)
• a __________________ group
• a nitrogenous base (adenine, thymine, cytosine,
  guanine, and uracil)
• Purines A G Pyrimidines C, U, T

11
DNA structure
Genetic structure/function

• 2 strands twisted into a double helix
• sugar -phosphate backbone
• nitrogenous bases form steps in ladder (inside
  helix)
• constancy of base pairing (purines pair with
  pyrimidines)
• _______________ with 2 hydrogen bonds
• (RNA A with U)
• __________________ with 3 hydrogen bonds
• antiparallel strands __________________
• each strand provides a template for the exact
  copying of a new strand
• order of bases constitutes the DNA code

12
Genetic structure/function
13
Genetic structure/function
14
Genetic structure/function
Torsion in helix and steps of nucleotides
results in major and minor grooves in helix
15
Significance of DNA structure
Genetic structure/function

• Maintenance of code during reproduction.
  Constancy of base pairing guarantees that the
  code will be retained.
• Providing variety. Order of bases responsible for
  unique qualities of each organism (gene sequence)
• Possible arrangement of nucleotides is nearly
  infinite 4n where nnucleotides. So for a 1000
  bp gene, 4n combinations 41000 or 1.5 x 10602
  !!

16
History of DNA structure
Genetics - History

• __________________________ 1944 showed DNA
  was the molecule carrying the blueprint for life.
  Won Nobel prize.
• Erwin Chargaff components of DNA
• Maurice Wilkins and ______________________ XRay
  crystallography gave clue to double helix
  structure
• Structural model credit (Nobel prize) goes to
  _________________________________- 1953

17
DNA replication
Genetic mechanisms -Replication

• Due to base pairing, each strand serves as
  template for the synthesis of a new strand
• DNA replication is ________________because each
  chromosome ends up with one new strand of DNA and
  one old strand.

DNA A with T, G with C so one strand can be
template for its complement strand
18
Semi-conservative replication of DNA
Genetic mechanisms
Genetic mechanisms -Replication
19
DNA replication (bacteria)
Genetic mechanisms -Replication

• Steps
• Begins at an origin of replication
• ___________unwinds and unzips the DNA double
  helix (replication fork)
• Strands are kept separate by SSBP (single strand
  binding proteins)
• An RNA ___________is synthesized (primase) and
  primes (DNA polymerase III cannot initiate
  synthesis on its own)

20
Replication (contd)
Genetic mechanisms -Replication

• DNA polymerase III adds nucleotides in a 5 to 3
  direction works on both strands at once (see
  movie!)
• Leading strand synthesized continuously in 5
  to 3 direction
• Lagging strand synthesized 5 to 3 in short
  segments (Okazaki fragments)
• overall direction of DNA pol III movement is 3
  to 5
• ___________ removes primers (RNA) and replaces
  with DNA
• ___________ fills in gaps/nicks

21
Enzymes involved in DNA replication (short list)
Genetic mechanisms -Replication
22
Bacterial replicon
Genetic mechanisms -Replication
(origin is A/T rich, easy to separate)
SEE THE TWO MOVIES ON THE WEB SITE!!
23
Genetic mechanisms -Replication

• Separation of daughter molecules occurs via a
  nick which is then repaired
• Two completed molecules will go to daughter cells
  (binary fission)

24
Other types of replication
Genetic mechanisms -Replication

• Eukaryotes similar to this but there are
  thousands of replicons acting simultaneously
  (replication bubbles)
• Rolling circle replication small circular
  genetic material (plasmids)

25
Flow of genetic information
26
Flow of genetic information

• What are the products that genes encode?
• Structural genes code for proteins
• Genes that code for RNA (they are not
  translated!)
• Regulatory genes that control the expression of
  other genes
• ___________ is an organisms genetic makeup
• ___________ is the physical trait that results
  from the expression of the organisms genes
• How are genes expressed?
• transcription and translation

27
Gene expression
Genetic mechanisms -Gene expression

• ________________ DNA template is used to
  synthesize RNA (transcript)
• ______________________is the enzyme responsible
• ________________making a protein using the
  information provided by messenger RNA (mRNA)
  involves decoding the mRNA
• occurs on ribosomes, and involves tRNA and amino
  acids
• Proteins end product of gene expression
• Please view transcription translation movies on
  the web page

28
DNA-protein relationship
Genetic mechanisms -Gene expression

• Each triplet of nucleotides (codon) specifies a
  particular amino acid.
• structure ? function A proteins primary
  structure determines its shape function.
• Proteins determine phenotype. Living things are
  what their proteins make them.
• DNA is mainly a blueprint that tells the cell
  which kinds of proteins to make and how to make
  them.

29
?DNA-protein relationship
Genetic mechanisms -Gene expression
30
RNA
Genetic mechanisms -Gene expression

• Three types
• messenger RNA (mRNA)
• transfer RNA (tRNA)
• ribosomal RNA (rRNA)
• How does RNA differ from DNA?
• Uses Uracil (U) instead of Thymine (T)
• Single stranded (except in some viruses)
• Ribose is the sugar

31
(No Transcript)
32
Genetic mechanisms -Gene expression
DNA
Transcription RNA polymerase
RNA
Translation ribosomes
PROTEINS
33
Transcription - steps
Genetic mechanisms -Gene expression

• RNA polymerase binds to promoter region upstream
  of the gene
• RNA polymerase adds nucleotides complementary to
  the template strand of a segment of DNA in the 5
  to 3 direction (downstream of promoter)
• Uracil is placed as adenines complement (U with
  A)
• At termination, RNA polymerase recognizes signals
  and releases the transcript
• 100-1,200 bases long

34
Transcription
Genetic mechanisms -Gene expression
35
Genetic mechanisms -Gene expression
DNA
Transcription RNA polymerase
RNA
Translation ribosomes
PROTEINS
36
Translation - intiation
Genetic mechanisms -Gene expression

• Ribosomes assemble on the ___________of a mRNA
  transcript
• Ribosome scans the mRNA until it reaches the
  start codon, usually _______(met)
• A tRNA molecule with the complementary anticodon
  and methionine amino acid enters the P site of
  the ribosome binds to the mRNA
• mRNA triplet code is translated into amino acids
  (elongation)

37
Translation
Genetic mechanisms -Gene expression
38
Genetic mechanisms -Gene expression
39
Genetic mechanisms -Gene expression
40
Translation elongation
Genetic mechanisms -Gene expression

• A second tRNA with the complementary anticodon
  fills the A site
• A peptide bond is formed
• The first tRNA is released and the ribosome
  slides down to the next codon (5?3 reading
  frame triplet).
• Another tRNA fills the A site a peptide bond is
  formed.
• This process continues until a stop codon is
  encountered.

41
(No Transcript)
42
Translation termination
Genetic mechanisms -Gene expression

• Termination (________) codons UAA, UAG, and UGA
  are codons for which there is no corresponding
  tRNA.
• When this codon is reached, the ribosome falls
  off and the last tRNA is removed from the
  polypeptide.

43
Polyribosomal complex transcription and
multiple translation simultaneously (bacteria)
44
Eucaryotic transcription translation differs
from procaryotic
Genetic mechanisms -Gene expression

• Do not occur simultaneously. Transcription occurs
  in the nucleus and translation occurs in the
  cytoplasm.
• Eucaryotic start codon is AUG, but it does not
  use formyl-methionine.
• Eucaryotic mRNA encodes a single protein, unlike
  bacterial mRNA which encodes many (operon).
• Eucaryotic DNA contains introns intervening
  sequences of noncoding DNA- which have to be
  spliced out of the final mRNA transcript.

45
Split gene of eucaryotes
Genetic mechanisms -Gene expression
46
Some transcribed genes arent translated
Genetic mechanisms -Gene expression

• Genes encoding for RNAs such as tRNA, rRNA, RNA
  primers (used in DNA replication)
• These RNAs have 2ndary structure like proteins
  but function as RNA

47
Regulation of protein synthesis metabolism
48
Operons
Gene regulation

• a coordinated set of genes, all of which are
  regulated as a single unit. Found in prokaryotes.
• 2 types based on regulation
• ________________ operon is turned ON by
  substrate catabolic operons- enzymes needed to
  metabolize a nutrient are produced when needed
• ________________ operon is turned OFF by the
  product synthesized anabolic operon enzymes
  used to synthesize an amino acid stop being
  produced when enough is made

49
Lactose operon inducible operon
Gene regulation

• Made of 3 segments
• ________________gene that codes for
  ________________
• ___________locus- composed of promoter and
  operator
• ___________locus- made of 3 genes each coding for
  an enzyme needed to catabolize lactose
• b-galactosidase hydolyzes lactose (gal and glu)
• permease - brings lactose across cell membrane
• b-galactoside transacetylase uncertain function

50
Lac operon inducible
Gene regulation

• ________________
• In the absence of lactose the repressor binds
  with the operator locus and blocks transcription
  of downstream structural genes
• Lactose ____________________________
• Binding of lactose to the repressor protein
  changes its shape and causes it to fall off the
  operator. RNA polymerase can bind to the
  promoter. Structural genes are transcribed.

51
Lactose operon
Gene regulation

• Repressor (protein) is a product of a regulator
  gene elsewhere in the genome
• It is attached to operator in absence of
  substrate and blocks transcription of genes
  downstream

• Lactose (substrate, inducer) binds repressor
• Repressor changes shape, comes off operator
• RNA pol can now transcribe gene
• Enzymes for lactose catabolism are translated
• When lactose levels go back down, repressor will
  bind to operator again

52
Arginine operon repressible
Gene regulation

• Normally on and will be turned off when product
  is no longer needed (excess).
• When excess arginine is present, it binds to the
  repressor and changes it. Then the repressor
  binds to the operator and blocks arginine
  synthesis.

53
Repressible operon

• Repressor isnt attached to operator, so operon
  transcription is continuous (genes are on) and
  ARG is being made
• This continues is as long as ARG is being used by
  the cell

• Excess product (ARG) builds up from not being
  used by cell
• ARG (corepressor) binds repressor which changes
  shape, and binds operator
• RNA pol cannot transcribe gene
• When ARG levels get too low, operator will fall
  off and transcription will begin again

54
Antibiotics that affect gene expression

• Rifamycin binds to RNA polymerase
• Actinomycin D - binds to DNA halts mRNA chain
  elongation
• Erythromycins interfere with attachment of mRNA
  to ribosomes
• Chloramphenicol, linomycin tetracycline - bind
  to ribosome and block elongation
• Streptomycin inhibits peptide initiation
  elongation
• Problem with drugs that affect prokaryotic
  ribosomes is that they affect ________________too

55
Changing the genetic code

• Mutations change in ______________ sequence
• ________________ addition of genes from an
  outside source (another cell, another organism)

56
Mutations

• Any permanent, inheritable change in genetic
  information
• Alteration of the nucleotide sequence (ATGC)
• Involves either loss, addition or rearrangement
  of base pairs
• Spontaneous mutation random, due to replication
  error
• Induced mutation result from exposure to
  mutagens (physical, chemical disrupt DNA)

57
(No Transcript)
58
Types of Mutations
mutations

• ________________mutation addition, deletion or
  substitution of a few bases
• ________________mutation causes change in a
  single amino acid
• ________________mutation changes a normal codon
  into a stop codon
• ________________mutation alters a base but does
  not change the amino acid

59
(No Transcript)
60
Excision repair
Mutations -repair
61
Ames Test
mutations
Also lacks DNA repair enzymes and has leaky cell
walls
Chemicals that produce an increased of back
mutations (more than spontaneous) are considered
mutagens
Spontaneous Back-mutation
Induced Back-mutation
62
Types of intermicrobial exchange
Genetic Recombination
Recombination events genetic transfer resulting
in a new strain different from both parent strains
63
Conjugation

• _____________ transfer of a plasmid or
  chromosomal fragment from a donor cell to a
  recipient cell via a direct connection
• Gram-negative cell donor has a fertility plasmid
  (F plasmid, F' factor) that allows the synthesis
  of a conjugative pilus
• Recipient cell is a related species or genus
  without a fertility plasmid
• Donor transfers fertility plasmid to recipient
  through pilus

63
64
Genetic Recombination -Conjugation
65
Figure 9.23 (2)
65
66
_____________

• High-frequency recombination donors fertility
  plasmid has been integrated into the bacterial
  chromosome
• When conjugation occurs, a portion of the
  chromosome and a portion of the fertility plasmid
  are transferred to the recipient

66
67
Figure 9.23 (3)
67
68
Transformation
Genetic Recombination -transformation

• _____________ chromosome fragments from a lysed
  cell are accepted by a recipient cell the
  genetic code of the DNA fragment is acquired by
  the recipient
• Donor and recipient cells can be unrelated
• Useful tool in recombinant DNA technology

69
Genetic Recombination -transformation
Figure 9.24
Insert figure 9.23 transformation
69
70
Consequences of changing the genetic code

• Anything that alters the DNA sequence (Mutations,
  transposons, transformation, etc.) can be
  beneficial or harmful to the microbe
• Examples
• Recombination events can result in increased
  fitness to the microbe, such as gaining
  antibiotic resistance, virulence factors etc.
• Some mutations or disruptions of genes
  (transposons) can be lethal to the cell if they
  interrupt crucial genes (like those involved with
  metabolism)
• Remember - Mutations cause harm if they change
  the final protein product to a non-functional
  protein. So if the AA is the same, the protein is
  not changed. (example AUG ? AUA Both are
  methionine) (Silent mutations)