1. Introduction to Microbiology Lecture 4
“Small creatures, big impacts”
Lecture 4

2. Introduction to Microbiology
Lecture outline
From DNA to proteins (continued)
Viruses
Molecular microbiology basics
Introduction to Microbiology

3. Short version- read book for more details
From DNA to proteins
Short version- read book for more details

4. Proteins (polypeptide): links between genotype and phenotype
Gene expression: process by which DNA directs protein synthesis
Transcription
Translation
From DNA to proteins

5. 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

6. DNA
TRANSCRIPTION
mRNA
From DNA to proteins
(a) Bacterial cell

7. From DNA to proteins DNA TRANSCRIPTION mRNA Ribosome TRANSLATION
Polypeptide
(a) Bacterial cell

8. From DNA to proteins Nuclear envelope DNA TRANSCRIPTION Pre-mRNA
(b) Eukaryotic cell

9. From DNA to proteins Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA
RNA PROCESSING
From DNA to proteins
mRNA
(b) Eukaryotic cell

10. From DNA to proteins Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA
RNA PROCESSING
From DNA to proteins
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell

11. 20 amino acids: but only four types of nucleotide bases in DNA
Triplet code: a series of non-overlapping, 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

12. Transcription: From DNA to proteins
one of the two DNA strands (“template strand”) provides a template for ordering the nucleotides in mRNA
From DNA to proteins

13. From DNA to proteins Gene 2 Gene 1 Gene 3 template strand Codon DNA
molecule
Gene 1
Gene 2
Gene 3
template
strand
TRANSCRIPTION
Codon
From DNA to proteins

14. Translation: From DNA to proteins
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

15. From DNA to proteins Gene 2 Gene 1 Gene 3 template strand mRNA Codon
molecule
Gene 1
Gene 2
Gene 3
template
strand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
From DNA to proteins

16. Genetic code: nearly universal (simplest bacteria to the most complex animals)
Second mRNA base
First mRNA base (5 end of codon)
Third mRNA base (3 end of codon)
From DNA to proteins

17. From DNA to proteins Codons must be read in the correct reading frame
mRNA
Correct
Incorrect –1 +1
From DNA to proteins

18. 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

19. 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

20. From DNA to proteins Amino acids tRNA with amino acid attached
Polypeptide
Ribosome
Amino
acids
tRNA with
amino acid
attached
tRNA
Anticodon
Trp
Phe
Gly
Codons 3 5
mRNA
From DNA to proteins

21. Viruses

22. Viruses Viruses: “a kind of borrowed life” Viruses are not cells
between life-forms and chemicals
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

23. Viruses Capsid: Protein shell that encloses the viral genome
Built from protein subunits called capsomeres
various structures
Viruses

24. Various shapes of Viruses
RNA
Capsomere
of capsid
DNA
Glycoprotein
18  250 nm
70–90 nm (diameter)
Glycoproteins
80–200 nm (diameter)
80  225 nm
Membranous
envelope
Capsid
Head
Tail
sheath
fiber
50 nm
20 nm
(a) Tobacco mosaic
virus
(b) Adenoviruses
(c) Influenza viruses
(d) Bacteriophage T4
Viruses

25. Viruses Bacteriophages Infect bacteria (e.g.Escherichia coli)
Takeover cellular machinery
Viruses

26. 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

27. 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 self-assemble into new viruses
Viruses

28. Viruses VIRUS Entry and uncoating DNA Capsid Transcription
and manufacture
of capsid proteins
Self-assembly of
new virus particles
and their exit from
the cell
Entry and
uncoating
VIRUS 1 2 3
DNA
Capsid 4
Replication
HOST CELL
Viral DNA
mRNA
proteins
Viruses

29. Molecular microbiology basics

30. 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 heat-tolerant bacteria, e.g. Thermus aquaticus
Chain reaction: produces exponentially growing population of identical DNA molecules
Molecular microbiology basics

31. molecules; 2 molecules (in white boxes) match target sequence5
Genomic DNA
TECHNIQUE
Cycle 1 yields 2
molecules
Denaturation
Annealing
Extension
Cycle 2 yields 4
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence
Target sequence
Primers
New nucleo- tides 3 1 3
Molecular microbiology basics

32. Molecular microbiology basics
DNA sequencing
Sequences of DNA fragments determined by dideoxy chain termination method
Modified nucleotides- dideoxyribonucleotides (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

33. Molecular microbiology basics
DNA (template strand)
TECHNIQUE
DNA polymerase
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Molecular microbiology basics

34. Molecular microbiology basics
TECHNIQUE
RESULTS
DNA (template strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Direction of movement of strands
Detector
Last base of longest labeled strand
Last base of shortest labeled strand
Molecular microbiology basics

35. 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

36. 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

37. Molecular microbiology basics
DNA of chromosome
Cell containing gene of interest
Gene inserted into plasmid
Plasmid put into bacterial cell
Recombinant DNA (plasmid)
Recombinant bacterium
Bacterial chromosome
Bacterium
Gene of interest
Plasmid 2 1
Molecular microbiology basics

38. Molecular microbiology basics
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Basic research and various applications
Copies of gene
Protein harvested
Basic research on gene
Basic research on protein 4
Recombinant bacterium
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth 3
Molecular microbiology basics

39. 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

40. 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

41. Molecular microbiology basics
FISH: Examples from my own research
Molecular microbiology basics