Introduction to Microbiology
“Small creatures, big impacts”
Lecture 4
Introduction to Microbiology
Lecture outline
• From DNA to proteins
(continued)
• Viruses
• Molecular microbiology basics
From DNA to proteins
Short version- read book for
more details
From DNA to proteins
• Proteins (polypeptide): links
between genotype and phenotype
• Gene expression: process by which
DNA directs protein synthesis
– Transcription
– Translation
From DNA to proteins
• RNA: intermediate between genes and
the proteins for which they code
• Transcription: synthesis of RNA under
the direction of DNA
– produces messenger RNA (mRNA)
• Translation: synthesis of a polypeptide,
which occurs under the direction of
mRNA
– Ribosomes: the sites of translation
From DNA to proteins
TRANSCRIPTION
DNA
mRNA
(a) Bacterial cell
From DNA to proteins
DNA
TRANSCRIPTION
mRNA
Ribosome
TRANSLATION
Polypeptide
(a) Bacterial cell
From DNA to proteins
Nuclear
envelope
TRANSCRIPTION
DNA
Pre-mRNA
(b) Eukaryotic cell
From DNA to proteins
Nuclear
envelope
TRANSCRIPTION
RNA PROCESSING
mRNA
(b) Eukaryotic cell
DNA
Pre-mRNA
From DNA to proteins
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell
From DNA to proteins
• 20 amino acids: but only four types
of nucleotide bases in DNA
• Triplet code: a series of nonoverlapping, three-nucleotide
words
– The smallest units of uniform length
that can code for all the amino acids
• Example: AGT on DNA  amino
acid serine in protein
From DNA to proteins
• Transcription:
–one of the two DNA strands
(“template strand”) provides a
template for ordering the
nucleotides in mRNA
From DNA to proteins
DNA
molecule
Gene 2
Gene 1
Gene 3
DNA
template
strand
TRANSCRIPTION
Codon
From DNA to proteins
• Translation:
–mRNA base triplets (“codons”)
are read in the 5 to 3 direction
–64 codons total
–61 code for amino acids; 3 are
“stop” signals to end translation
From DNA to proteins
DNA
molecule
Gene 2
Gene 1
Gene 3
DNA
template
strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Protein
Third mRNA base (3 end of codon)
Second mRNA base
First mRNA base (5 end of codon)
From DNA to proteins
Genetic code: nearly universal (simplest
bacteria to the most complex animals)
From DNA to proteins
Codons must be read in the correct reading frame
mRNA
Correct
0
Incorrect
–1
Incorrect
+1
From DNA to proteins
• Transfer RNA (tRNA)
– Helps cell translates an mRNA
message into protein
– Many types
– Each carries a specific amino acid on
one end
– Each has an anticodon on the other
end; the anticodon base-pairs with a
complementary codon on mRNA
From DNA to proteins
• Ribosomes
– Facilitate specific coupling of tRNA
anticodons with mRNA codons in
protein synthesis
– Two ribosomal subunits (large and
small)
– Made of proteins and ribosomal RNA
(rRNA)
From DNA to proteins
Amino
acids
Polypeptide
tRNA with
amino acid
attached
Ribosome
tRNA
Anticodon
Codons
5
mRNA
3
Viruses
• Viruses: “a kind of borrowed life”
– between life-forms and chemicals
Viruses
• Viruses are not cells
– Very small infectious particles
– Nucleic acid enclosed in a protein coat
– In some cases, a membranous envelope
• Viral genomes:
– Double- or single-stranded DNA (DNA virus)
– Double- or single-stranded RNA (RNA virus)
Viruses
• Capsid:
–Protein shell that encloses the
viral genome
–Built from protein subunits called
capsomeres
–various structures
Various shapes of Viruses
RNA
Capsomere
DNA
Membranous
envelope
RNA
Head
DNA
Viruses
Capsid
Capsomere
of capsid
18
Tail
fiber
Glycoprotein Glycoproteins
250 nm
70–90 nm (diameter) 80–200 nm (diameter)
20 nm
Tail
sheath
50 nm
50 nm
80
225 nm
50 nm
(a) Tobacco mosaic (b) Adenoviruses (c) Influenza viruses (d) Bacteriophage T4
virus
• Bacteriophages
Viruses
– Infect bacteria (e.g.Escherichia coli)
– Takeover cellular machinery
Viruses
• Viruses: obligate intracellular
parasites
–can reproduce only within a host
cell
• Each virus has a host range
–limited number of host cells that
it can infect
Viruses
Steps of a Viral infection:
• Virus attaches to cell and inserts DNA
• Cell begins to manufacture viral proteins
• Virus uses host enzymes, ribosomes,
tRNAs, amino acids, ATP, and other
molecules
• Viral nucleic acid molecules and
capsomeres spontaneously selfassemble into new viruses
VIRUS
1 Entry and
DNA
uncoating
Capsid
3 Transcription
and manufacture
of capsid proteins
2 Replication
HOST CELL
Viruses
Viral DNA
mRNA
Viral DNA
Capsid
proteins
4 Self-assembly of
new virus particles
and their exit from
the cell
Molecular microbiology basics
Molecular microbiology basics
Polymerase Chain Reaction (PCR)
• Can produce many copies of a specific
segment of DNA
• Three-step cycle—heating, cooling, and
replication
• Uses DNA polymerase from heattolerant bacteria, e.g. Thermus
aquaticus
• Chain reaction: produces exponentially
growing population of identical DNA
molecules
Molecular microbiology basics
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Molecular microbiology basics
DNA sequencing
• Sequences of DNA fragments determined
by dideoxy chain termination method
• Modified nucleotidesdideoxyribonucleotides (ddNTP) attach to
synthesized DNA strands of different
lengths
• Each type of ddNTP tagged with distinct
fluorescent label
• DNA sequence read from spectrogram
Molecular microbiology basics
TECHNIQUE
DNA
(template strand)
Primer
DNA
polymerase
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
Molecular microbiology basics
TECHNIQUE
DNA (template
strand)
Labeled strands
Shortest
Direction
of movement
of strands
Longest
Longest labeled strand
Detector
Laser
RESULTS
Shortest labeled strand
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Molecular microbiology basics
DNA cloning
• Cloning= making copy of segment of DNA
– Sequencing
– Expression into proteins
• Most methods use bacteria and plasmids
• Plasmids: small circular DNA molecules
that replicate separately from the bacterial
chromosome
Molecular microbiology basics
Steps of DNA cloning
• Foreign DNA inserted into plasmid,
• New plasmid, called recombinant
plasmid inserted into bacterial cell
• Reproduction in bacterial cell: many
copies of plasmid including foreign
DNA
• Production of multiple copies of a
single gene!
Molecular microbiology basics
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
Molecular microbiology basics
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Protein harvested
4 Basic research and
Basic
research
on gene
Gene for pest
resistance inserted
into plants
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Molecular microbiology basics
Microbial diversity studies
• DNA purified from environment,
e.g. soil
• Cloned and sequenced
• Most common type of DNA
sequence: ribosomal DNA (rDNA)
Molecular microbiology basics
Fluorescent in situ hybridization
(FISH)
•Probe: small sequence (approx. 25 bases)
complementary to rDNA, attached to
fluorescent molecule
•Apply probe to whole microbial cells
(“fixed” with Ethanol)
•Probe binds to ribosomal RNA
•View using fluorescence microscopy- only
cells that contain complementary
sequence will fluoresce
Molecular microbiology basics
FISH: Examples from my own research