Microbial Molecular Biology

1
Microbial Molecular Biology
2
Counting Bacteria from todays paper
Levels over 100,000/ml are considered a
significant health hazard by the WHO
3
Counting Bacteria
4

• Central dogma of molecular biology
• DNA to RNA to protein

5
Genetic Information

• Genetic information flow can be divided into
  three stages
• Replication DNA is duplicated
• Transcription information from DNA is
  transferred to RNA
• mRNA (messenger RNA) encodes polypeptides
• tRNA (transfer RNA) plays role in protein
  synthesis
• rRNA (ribosomal RNA) plays role in protein
  synthesis
• Translation information in mRNA is used to build
  polypeptides

6
Synthesis of the Informational Macromolecules
7
Macromolecules and Genetic Information

• Central dogma of molecular biology
• DNA to RNA to protein
• Eukaryotes each gene is transcribed individually
• Prokaryotes multiple genes may be transcribed
  together

8
Transcription in Prokaryotes
Figure 7.2
9
DNA Structure (overview slide)

• Double Helix
• Complementary base pairing
• Antiparallel
• Supercoiling
• Chromosomes and Other Genetic Elements

10
The Double Helix

• Four nucelotides found in DNA
• Adenine (A)
• Guanine (G)
• Cytosine (C)
• Thymine (T)
• Backbone of DNA chain is alternating phosphates
  and the pentose sugar deoxyribose
• Phosphates connect 3'- carbon of one sugar to 5?
  of the adjacent sugar

11
The Double Helix

• All cells and some viruses have DNA in double-
  stranded molecule
• Two strands are antiparallel
• Two strands have complementary base sequences
• Adenine always pairs with Thymine
• Guanine always pairs with Cytosine
• Two strands form a double helix

12
DNA Structure
13
Genome - Genetic Elements

• Genome entire complement of genes in cell or
  virus
• Chromosome main genetic element in prokaryotes
• Other genetic elements include virus genomes,
  plasmids, organellar genomes, and transposable
  elements

14
Genetic Elements

• Viruses contain either RNA or DNA genomes
• Can be linear or circular
• Can be single or double stranded
• Plasmids replicate separately from chromosome
• Great majority are double stranded
• Circular (most)
• Generally beneficial for the cell (i.e.,
  antibiotic resistance)
• NOT extracellular, unlike viruses
• Plasmid is a genetic element that is expendable
  and rarely contains genes needed for growth under
  all conditions

15
Chromosomes and Other Genetic Elements

• Transposable Elements
• Segment of DNA that can move from one site to
  another site on the same or different DNA
  molecule
• Inserted into other DNA molecules
• Insertion sequences
• Transposons

16
Kinds of Genetic Elements
17
DNA replication

• Origin of replication
• Where on the DNA molecule replication begins

18
Replication of Circular DNA The Theta Structure
Figure 7.16
19
DNA replication

• DNA replication is semiconservative
• Each of the two progeny double helices have one
  parental and one new strand
• Replication ALWAYS proceeds from the 5' end to
  the 3' end

20
Overview of DNA Replication
DNA Replication-overview Animation link
21
Structure of the DNA chain and Mechanism of Growth
Figure 7.11
22
Templates and Enzymes

• DNA polymerases catalyze the addition of
  nucleotides
• DNA polymerases require a primer
• Primer made from RNA by an enzyme Primase

23
The Replication Fork

• DNA synthesis begins at the origin of replication
  in prokaryotes
• Replication fork zone of unwound DNA where
  replication occurs
• DNA helicase unwinds the DNA
• Extension of DNA
• Occurs continuously on the leading strand
• Discontinuously on the lagging strand
• Okazaki fragments are on lagging strand

24
Events at the DNA Replication Fork
Figure 7.13
25
Sealing Two Fragments on the Lagging Strand
DNA Replication Tutorial link
26
Proofreading

• DNA replication is extremely accurate
• Proofreading helps to ensure high fidelity
• Mutation rates in cells are 10-810-11 errors per
  base inserted
• Polymerase can detect mismatch through incorrect
  hydrogen bonding

27
Overview of Transcription
Transcription overview animation link

• Transcription (DNA to RNA) is carried out by RNA
  polymerase
• RNA polymerase uses DNA as template
• RNA precursors are ATP, GTP, CTP, and UTP
• Chain growth is 5' to 3' just like DNA replication

28
Overview of Transcription

• Only one of the two strands of DNA are
  transcribed by RNA polymerase for any gene
• Genes are present on both strands of DNA, but at
  different locations
• RNA polymerase has five different subunits
• RNA polymerase recognizes DNA sites called
  promoters

29
Overview of Transcription

• Promoters site of initiation of transcription
• Promoters are recognized by sigma factor of RNA
  polymerase
• Transcription stops at specific sites called
  transcription terminators
• Unlike DNA replication, transcription involves
  smaller units of DNA
• Often as small as a single gene
• Allows cell to transcribe different genes at
  different rates

30
Transcription Steps in RNA Synthesis
Figure 7.21a
31
Sigma Factors and Consensus Sequences

• Sigma factors recognize two highly conserved
  regions of promoter
• Two regions within promoters are highly conserved
• Pribnow box located 10 bases before the start of
  transcription (-10 region)
• -35 region located 35 bases upstream of
  transcription

32
The Interaction of RNA Polymerase with the
Promoter
Figure 7.22
33
Termination of Transcription

• Termination of RNA synthesis is governed by a
  specific DNA sequence

Transcription tutorial animation link
34
The Unit of Transcription

• Unit of transcription unit of chromosome bounded
  by sites where transcription of DNA to RNA is
  initiated and terminated
• Most genes encode proteins, but some RNAs are not
  translated (i.e., rRNA, tRNA)
• mRNA have short half-lives (a few minutes)

35
The Unit of Transcription

• Prokaryotes often have genes related to the same
  process clustered together
• These genes are transcribed all at once as a
  single mRNA
• An mRNA encoding a group of cotranscribed genes
  is called a polycistronic mRNA
• Operon a group of related genes cotranscribed on
  a polycistronic mRNA
• Allows for expression of multiple genes to be
  coordinately regulated

36
The Genetic Code

• Translation the synthesis of proteins from RNA
• Genetic code a triplet of nucleic acid bases
  (codon) encodes a single amino acid
• Specific codons for starting and stopping
  translation
• Degenerate code multiple codons encode a single
  amino acid
• Anti-codon on tRNA recognizes codon

37
The Genetic Code as Expressed by Triplet Bases of
mRNA
38
The Genetic Code

• Stop codons signal the termination of
  translation (UAA, UAG, and UGA)
• Start Codon translation begins with AUG
• Reading frame triplet code requires translation
  to begin at the correct nucleotide
• Open Reading Frame (ORF) AUG followed by a
  number of codons and a stop codon in the same
  reading frame

39
Possible Reading Frames in an mRNA
Figure 7.26
40
Transfer RNA

• Transfer RNA at least one tRNA per amino acid
• Specific for both a codon and its particular
  amino acid
• tRNA is cloverleaf in shape
• Anti-codon three bases of tRNA that recognize
  three complementary bases on mRNA

41
Translation The Process of Protein Synthesis
Translation-overview animation link

• Ribosomes sites of protein synthesis
• Thousands of ribosomes per cell
• Combination of rRNA and protein

42
Translation The Process of Protein Synthesis

• Translation is broken down into three main steps
  
• 1) Initiation two ribosomal subunits assemble
  with mRNA
• Begins at an AUG start codon
• 2) Elongation amino acids are brought to the
  ribosome and are added to the growing polypeptide
• Occurs in the A and P sites of ribosome
• Translocation movement of the tRNA holding the
  polypeptide from the A to the P site
• Polysomes a complex formed by ribosomes
  simultaneously translating mRNA

43
The Ribosome and Protein Synthesis
Figure 7.29a
44
The Ribosome and Protein Synthesis
45
Translation The Process of Protein Synthesis

• Steps of Translation (contd)
• 3) Termination occurs when ribosome reaches a
  stop codon
• Release factors (RF) recognize stop codon and
  cleave polypeptide from tRNA
• Ribosome subunits then dissociate
• Subunits free to form new initiation complex and
  repeat process

Translation-indepth animation link
46
Translation The Process of Protein Synthesis

• Many antibiotics inhibit translation by
  interacting with ribosomes
• Streptomycin, chloramphenicol, tetracycline, etc.