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Michael Philips

Michael Philips

Gene Regulation in Prokaryotes

Slide Duration:

Table of Contents

I. The Beginnings of Molecular Biology
Biochemistry Review: Importance of Chemical Bonds

53m 29s

Intro
0:00
Lesson Overview
0:14
Chemical Bonds
0:41
Attractive Forces That Hold Atoms Together
0:44
Types of Bonds
0:56
Covalent Bonds
1:34
Valence Number
1:58
H O N C P S Example
2:50
Polar Bonds
7:23
Non-Polar Bond
8:46
Non-Covalent Bonds
9:46
Ionic Bonds
10:25
Hydrogen Bonds
10:52
Hydrophobic Interactions
11:34
Van Der Waals Forces
11:58
Example 1
12:51
Properties of Water
18:27
Polar Molecule
13:34
H-bonding Between Water H20 Molecules
19:29
Hydrophobic Interactions
20:30
Chemical Reactions and Free Energy
22:52
Transition State
23:00
What Affect the Rate
23:27
Forward and Reserve Reactions Occur Simultaneously But at Different Rate
23:51
Equilibrium State
24:29
Equilibrium Constant
25:18
Example 2
26:16
Chemical Reactions and Free Energy
27:49
Activation Energy
28:00
Energy Barrier
28:22
Enzymes Accelerate Reactions by Decreasing the Activation Energy
29:04
Enzymes Do Not Affect the Reaction Equilibrium or the Change in Free Energy
29:22
Gibbs Free Energy Change
30:50
Spontaneity
31:18
Gibbs Free Energy Change Determines Final Concentrations of Reactants
34:36
Endodermic vs. Exothermic Graph
35:00
Example 3
38:46
Properties of DNA
39:37
Antiparallel Orientation
40:29
Purine Bases Always Pairs Pyrimidine Bases
41:15
Structure Images
42:36
A, B, Z Forms
43:33
Major and Minor Grooves
44:09
Hydrogen Bonding and Hydrophobic Interactions Hold the Two Strands Together
44:39
Denaturation and Renaturation of DNA
44:56
Ways to Denature dsDNA
45:28
Renature When Environment is Brought Back to Normal
46:05
Hyperchromiicity
46:36
Absorbs UV Light
47:01
Spectrophotometer
48:01
Graph Example?
49:05
Example 4
51:02
Mendelian Genetics & Foundational Experiments

1h 9m 27s

Intro
0:00
Lesson Overview
0:22
Gregor Johann Mendel
1:01
Was a Biologist and Botanist
1:14
Published Seminal Paper on Hybridization and Inheritance in the Pea Plant
1:20
Results Criticized
1:28
Father of Modern Genetics
1:59
Mendel’s Laws
2:19
1st Law: Principle of Independent Segregation of Alleles
2:27
2nd Law: Principle of Independent Assortment of Genes
2:34
Principle of Independent Segregation (of Alleles)
2:41
True Breeding Lines / Homozygous
2:42
Individuals Phenotypes Determined by Genes
3:15
Alleles
3:37
Alleles Can Be Dominant or Recessive
3:50
Genotypes Can be Experimentally Determined by Mating and Analyzing the Progeny
5:36
Individual Alleles Segregate Independently Into Gametes
5:55
Example 1
6:18
Principle of Independent Segregation (of Alleles)
16:11
Individual Genes Sort Independently Into Gametes
16:22
Each Gamete Receives One Allele of Each Gene: 50/50 Chance
16:46
Genes Act Independently to Determine Unrelated Phenotypes
16:57
Example: Punnett Square
17:15
Example 2
21:36
The Chromosomal Theory of Inheritance
30:41
Walter S Sutton Linked Cytological Studies with Mendels Work
31:02
Diploid Cells Have Two Morphologically Similar Sets of Chromosomes and Each Haploid Gamete Receives One Set
31:17
Genes Are on Chromosome
31:33
Gene for Seed Color’s on a Different Chromosome Than Gene for Seed Texture
31:44
Gene Linkage
31:55
Mendel’s 2nd Law
31:57
Genes Said to Be Linked To Each Other
32:09
Linkage Between Genes
32:29
Linkage is Never 100% Complete
32:41
Genes are Found on Chromosomes
33:00
Thomas Hunt Morgan and Drosophila Melanogaster
33:01
Mutation Linked to X Chromosome
33:15
Linkage of White Gene
33:23
Eye Color of Progeny Depended on Sex of Parent
33:34
Y Chromosome Does Not Carry Copy of White Gene
33:44
X Linked Genes, Allele is Expressed in Males
33:56
Example
34:11
Example 3
35:52
Discovery of the Genetic Material of the Cell
41:52
Transforming Principle
42:44
Experiment with Streptococcus Pneumoniae
42:55
Beadle and Tatum Proposed Genes Direct the Synthesis of Enzymes
45:15
One Gene One Enzyme Hypothesis
45:46
One Gene One Polypeptide Theory
45:52
Showing the Transforming Material was DNA
46:14
Did This by Fractionating Heat-Killed “S” Strains into DNA, RNA, and Protein
46:32
Result: Only the DNA Fraction Could Transform
47:15
Leven: Tetranucleotide Hypothesis
48:00
Chargaff Showed This Was Not the Case
48:48
Chargaff: DNA of Different Species Have Different Nucleotide Composition
49:02
Hershey and Chase: DNA is the Genetic Material
50:02
Incorporate Sulfur into Protein and Phosphorous into DNA
51:12
Results: Phosphorase Entered Bacteria and Progeny Phage, But no Sulfur
53:11
Rosalind Franklin’s “Photo 51” Showing the Diffraction Pattern of DNA
53:50
Watson and Crick: Double Helical Structure of DNA
54:57
Example 4
56:56
Discovery of the Genetic Material of the Cell
58:09
Kornberg: DNA Polymerase I
58:10
Three Postulated Methods of DNA Replication
59:22
Meselson and Stahl: DNA Replication is Semi-Conservative
1:00:21
How DNA Was Made Denser
1:00:52
Discovery of RNA
1:03:32
Ribosomal RNA
1:03:48
Transfer RNA
1:04:00
Messenger RNA
1:04:30
The Central Dogma of Molecular Biology
1:04:49
DNA and Replication
1:05:08
DNA and Transcription = RNA
1:05:26
RNA and Translation = Protein
1:05:41
Reverse Transcription
1:06:08
Cracking the Genetic Code
1:06:58
What is the Genetic Code?
1:07:04
Nirenberg Discovered the First DNA Triplet That Would Make an Amino Acid
1:07:16
Code Finished in 1966 and There Are 64 Possibilities or Triplet Repeats/ Codons
1:07:54
Degeneracy of the Code
1:08:53
II. Structure of Macromolecules
Structure of Proteins

49m 44s

Intro
0:00
Lesson Overview
0:10
Amino Acids
0:47
Structure
0:55
Acid Association Constant
1:55
Amino Acids Make Up Proteins
2:15
Table of 21 Amino Acid Found in Proteins
3:34
Ionization
5:55
Cation
6:08
Zwitterion
7:51
Anion
9:15
Example 1
10:53
Amino Acids
13:11
L Alpha Amino Acids
13:19
Only L Amino Acids Become Incorporated into Proteins
13:28
Example 2
13:46
Amino Acids
18:20
Non-Polar
18:41
Polar
18:58
Hydroxyl
19:52
Sulfhydryl
20:21
Glycoproteins
20:41
Pyrrolidine
21:30
Peptide (Amide) Bonds
22:18
Levels of Organization
23:35
Primary Structure
23:54
Secondary Structure
24:22
Tertiary Structure
24:58
Quaternary Structure
25:27
Primary Structure: Specific Amino Acid Sequence
25:54
Example 3
27:30
Levels of Organization
29:31
Secondary Structure: Local 3D
29:32
Example 4
30:37
Levels of Organization
32:59
Tertiary Structure: Total 3D Structure of Protein
33:00
Quaternary Structure: More Than One Subunit
34:14
Example 5
34:52
Protein Folding
37:04
Post-Translational Modifications
38:21
Can Alter a Protein After It Leaves the Ribosome
38:33
Regulate Activity, Localization and Interaction with Other Molecules
38:52
Common Types of PTM
39:08
Protein Classification
40:22
Ligand Binding, Enzyme, DNA or RNA Binding
40:36
All Other Functions
40:53
Some Functions: Contraction, Transport, Hormones, Storage
41:34
Enzymes as Biological Catalysts
41:58
Most Metabolic Processes Require Catalysts
42:00
Most Biological Catalysts Are Proteins
43:13
Enzymes Have Specificity of Reactants
43:33
Enzymes Have an Optimum pH and Temperature
44:31
Example 6
45:08
Structure of Nucleic Acids

1h 2m 10s

Intro
0:00
Lesson Overview
0:06
Nucleic Acids
0:26
Biopolymers Essential for All Known Forms of Life That Are Composed of Nucleotides
0:27
Nucleotides Are Composed of These
1:17
Nucleic Acids Are Bound Inside Cells
2:10
Nitrogen Bases
2:49
Purines
3:01
Adenine
3:10
Guanine
3:20
Pyrimidines
3:54
Cytosine
4:25
Thymine
4:33
Uracil
4:42
Pentoses
6:23
Ribose
6:45
2' Deoxyribose
6:59
Nucleotides
8:43
Nucleoside
8:56
Nucleotide
9:16
Example 1
10:23
Polynucleotide Chains
12:18
What RNA and DNA Are Composed of
12:37
Hydrogen Bonding in DNA Structure
13:55
Ribose and 2! Deoxyribose
14:14
DNA Grooves
14:28
Major Groove
14:46
Minor Groove
15:00
Example 2
15:20
Properties of DNA
24:15
Antiparallel Orientation
24:25
Phosphodiester Linkage
24:50
Phosphate and Hydroxyl Group
25:05
Purine Bases Always Pairs Pyramidine Bases
25:30
A, B, Z Forms
25:55
Major and Minor Grooves
26:24
Hydrogen Bonding and Hydrophobic Interactions Hold Strands Together
26:34
DNA Topology - Linking Number
27:14
Linking Number
27:31
Twist
27:57
Writhe
28:31
DNA Topology - Supercoiling
31:50
Example 3
33:16
III. Maintenance of the Genome
Genome Organization: Chromatin & Nucleosomes

57m 2s

Intro
0:00
Lesson Overview
0:09
Quick Glossary
0:24
DNA
0:29
Gene
0:34
Nucleosome
0:47
Chromatin
1:07
Chromosome
1:19
Genome
1:30
Genome Organization
1:38
Physically Cellular Differences
3:09
Eukaryotes
3:18
Prokaryotes, Viruses, Proteins, Small Molecules, Atoms
4:06
Genome Variance
4:27
Humans
4:52
Junk DNA
5:10
Genes Compose Less Than 40% of DNA
6:03
Chart
6:26
Example 1
8:32
Chromosome Variance - Size, Number, and Density
10:27
Chromosome
10:47
Graph of Human Chromosomes
10:58
Eukaryotic Cell Cycle
12:07
Requirements for Proper Chromosome Duplication and Segregation
13:07
Centromeres and Telomeres
13:28
Origins of Replication
13:38
Illustration: Chromosome
13:44
Chromosome Condensation
15:52
Naked DNA to Start
16:00
Beads on a String
16:13
Mitosis
16:52
Start with Two Different Chromosomes
17:18
Split Into Two Diploid Cells
17:26
Prophase
17:42
Prometaphase
17:52
Metaphase
19:10
Anaphase
19:27
Telophase
20:11
Cytokinesis
20:31
Cohesin and Condensis
21:06
Illustration: Cohesin and Condensis
21:19
Cohesin
21:38
Condensin
21:43
Illustration of What Happens
21:50
Cohesins
27:23
Loaded During Replication and Cleaved During Mitosis
27:30
Separase
27:36
Nucleosomes
27:59
Histone Core
28:50
Eight Histone Proteins
28:57
Octamer of Core Histones Picture
29:14
Chromosome Condensation via H1
30:59
Allows Transition to Compact DNA
31:09
When Not in Mitosis
31:37
Histones Decrease Available Binding Sites
32:38
Histone Tails
33:21
Histone Code
35:32
Epigenetic Code
35:56
Phosphorylation
36:45
Acetylation
36:57
Methylation
37:01
Ubiquitnation
37:04
Example 2
38:48
Nucleosome Assembly
41:22
Duplication of DNA Requires Duplication of Histones
41:50
Old Histones Are Recycled
42:00
Parental H3-H4 Tetramers Facilitate the Inheritance of Chromatin States
44:04
Example 3
46:00
Chromatin Remodeling
48:12
Example 4
53:28
DNA Replication

1h 9m 55s

Intro
0:00
Lesson Overview
0:06
Eukaryotic Cell Cycle
0:50
G1 Growth Phase
0:57
S Phase: DNA & Replication
1:09
G2 Growth Phase
1:28
Mitosis
1:36
Normal Human Cell Divides About Every 24 Hours
1:40
Eukaryotic DNA Replication
2:04
Watson and Crick
2:05
Specific Base Pairing
2:37
DNA Looked Like Tetrinucleotide
2:55
What DNA Looks Like Now
3:18
Eukaryotic DNA Replication - Initiation
3:44
Initiation of Replication
3:53
Primer Template Junction
4:25
Origin Recognition Complex
7:00
Complex of Proteins That Recognize the Proper DNA Sequence for Initiation of Replication
7:35
Prokaryotic Replication
7:56
Illustration
8:54
DNA Helicases (MCM 2-7)
11:53
Eukaryotic DNA Replication
14:36
Single-Stranded DNA Binding Proteins
14:59
Supercoils
16:30
Topoisomerases
17:35
Illustration with Helicase
19:05
Synthesis of the RNA Primer by DNA Polymerase Alpha
20:21
Subunit: Primase RNA Polymerase That Synthesizes the RNA Primer De Navo
20:38
Polymerase Alpha-DNA Polymerase
21:01
Illustration of Primase Function Catalyzed by DnaG in Prokaryotes
21:22
Recap
24:02
Eukaryotic DNA Replication - Leading Strand
25:02
Synthesized by DNA Polymerase Epsilon
25:08
Proof Reading
25:26
Processivity Increased by Association with PCNA
25:47
What is Processivity?
26:19
Illustration: Write It Out
27:03
The Lagging Strand/ Discontinuing Strand
30:52
Example 1
31:57
Eukaryotic DNA Replication - Lagging Strand
32:46
Discontinuous
32:55
DNA Polymerase Delta
33:15
Okazaki Fragments
33:36
Illustration
33:55
Eukaryotic DNA Replication - Okazaki Fragment Processing
38:26
Illustration
38:44
When Does Okazaki Fragments Happen
40:32
Okazaki Fragments Processing
40:41
Illustration with Okazaki Fragments Process Happening
41:13
Example 2
47:42
Example 3
49:20
Telomeres
56:01
Region of Repetitive Nucleotide Sequences
56:26
Telomeres Act as Chromosome Caps by Binding Proteins
57:42
Telomeres and the End Replication Problem
59:56
Need to Use a Primer
59:57
DNA Mutations & Repairs

1h 13m 8s

Intro
0:00
Lesson Overview
0:06
Damage vs. Mutation
0:40
DNA Damage-Alteration of the Chemical Structure of DNA
0:45
DNA Mutation-Permanent Change of the Nucleotide Sequence
1:01
Insertions or Deletions (INDELS)
1:22
Classes of DNA Mutations
1:50
Spontaneous Mutations
2:00
Induced Mutations
2:33
Spontaneous Mutations
3:21
Tautomerism
3:28
Depurination
4:09
Deamination
4:30
Slippage
5:44
Induced Mutations - Causes
6:17
Chemicals
6:24
Radiation
7:46
Example 1
8:30
DNA Mutations - Tobacco Smoke
9:59
Covalent Adduct Between DNA and Benzopyrene
10:02
Benzopyrene
10:20
DNA Mutations - UV Damage
12:16
Oxidative Damage from UVA
12:30
Thymidine Dimer
12:34
Example 2
13:33
DNA Mutations - Diseases
17:25
DNA Repair
18:28
Mismatch Repair
19:15
How to Recognize Which is the Error: Recognize Parental Strand
22:23
Example 3
26:54
DNA Repair
32:45
Damage Reversal
32:46
Base-Excision Repair (BER)
34:31
Example 4
36:09
DNA Repair
45:43
Nucleotide Excision Repair (NER)
45:48
Nucleotide Excision Repair (NER) - E.coli
47:51
Nucleotide Excision Repair (NER) - Eukaryotes
50:29
Global Genome NER
50:47
Transcription Coupled NER
51:01
Comparing MMR and NER
51:58
Translesion Synthesis (TLS)
54:40
Not Really a DNA Repair Process, More of a Damage Tolerance Mechanism
54:50
Allows Replication Past DNA Lesions by Polymerase Switching
55:20
Uses Low Fidelity Polymerases
56:27
Steps of TLS
57:47
DNA Repair
1:00:37
Recombinational Repair
1:00:54
Caused By Ionizing Radiation
1:00:59
Repaired By Three Mechanisms
1:01:16
Form Rarely But Catastrophic If Not Repaired
1:01:42
Non-homologous End Joining Does Not Require Homology To Repair the DSB
1:03:42
Alternative End Joining
1:05:07
Homologous Recombination
1:07:41
Example 5
1:09:37
Homologous Recombination & Site-Specific Recombination of DNA

1h 14m 27s

Intro
0:00
Lesson Overview
0:16
Homologous Recombination
0:49
Genetic Recombination in Which Nucleotide Sequences Are Exchanged Between Two Similar or Identical Molecules of DNA
0:57
Produces New Combinations of DNA Sequences During Meiosis
1:13
Used in Horizontal Gene Transfer
1:19
Non-Crossover Products
1:48
Repairs Double Strand Breaks During S/Gs
2:08
MRN Complex Binds to DNA
3:17
Prime Resection
3:30
Other Proteins Bind
3:40
Homology Searching and subsequent Strand Invasion by the Filament into DNA Duplex
3:59
Holliday Junction
4:47
DSBR and SDSA
5:44
Double-Strand Break Repair Pathway- Double Holliday Junction Model
6:02
DSBR Pathway is Unique
6:11
Converted Into Recombination Products by Endonucleases
6:24
Crossover
6:39
Example 1
7:01
Example 2
8:48
Double-Strand Break Repair Pathway- Synthesis Dependent Strand Annealing
32:02
Homologous Recombination via the SDSA Pathway
32:20
Results in Non-Crossover Products
32:26
Holliday Junction is Resolved via Branch Migration
32:43
Example 3
34:01
Homologous Recombination - Single Strand Annealing
42:36
SSA Pathway of HR Repairs Double-Strand Breaks Between Two Repeat Sequences
42:37
Does Not Require a Separate Similar or Identical Molecule of DNA
43:04
Only Requires a Single DNA Duplex
43:25
Considered Mutagenic Since It Results in Large Deletions of DNA
43:42
Coated with RPA Protein
43:58
Rad52 Binds Each of the Repeated Sequences
44:28
Leftover Non-Homologous Flaps Are Cut Away
44:37
New DNA Synthesis Fills in Any Gaps
44:46
DNA Between the Repeats is Always Lost
44:55
Example 4
45:07
Homologous Recombination - Break Induced Replication
51:25
BIR Pathway Repairs DSBs Encountered at Replication Forks
51:34
Exact Mechanisms of the BIR Pathway Remain Unclear
51:49
The BIR Pathway Can Also Help to Maintain the Length of Telomeres
52:09
Meiotic Recombination
52:24
Homologous Recombination is Required for Proper Chromosome Alignment and Segregation
52:25
Double HJs are Always Resolved as Crossovers
52:42
Illustration
52:51
Spo11 Makes a Targeted DSB at Recombination Hotspots
56:30
Resection by MRN Complex
57:01
Rad51 and Dmc1 Coat ssDNA and Promote Strand Invasion and Holliday Junction Formation
57:04
Holliday Junction Migration Can Result in Heteroduplex DNA Containing One or More Mismatches
57:22
Gene Conversion May Result in Non-Mendelian Segregation
57:36
Double-Strand Break Repair in Prokaryotes - RecBCD Pathway
58:04
RecBCD Binds to and Unwinds a Double Stranded DNA
58:32
Two Tail Results Anneal to Produce a Second ssDNA Loop
58:55
Chi Hotspot Sequence
59:40
Unwind Further to Produce Long 3 Prime with Chi Sequence
59:54
RecBCD Disassemble
1:00:23
RecA Promotes Strand Invasion - Homologous Duplex
1:00:36
Holliday Junction
1:00:50
Comparison of Prokaryotic and Eukaryotic Recombination
1:01:49
Site-Specific Recombination
1:02:41
Conservative Site-Specific Recombination
1:03:10
Transposition
1:03:46
Transposons
1:04:12
Transposases Cleave Both Ends of the Transposon in Original Site and Catalyze Integration Into a Random Target Site
1:04:21
Cut and Paste
1:04:37
Copy and Paste
1:05:36
More Than 40% of Entire Human Genome is Composed of Repeated Sequences
1:06:15
Example 5
1:07:14
IV. Gene Expression
Transcription

1h 19m 28s

Intro
0:00
Lesson Overview
0:07
Eukaryotic Transcription
0:27
Process of Making RNA from DNA
0:33
First Step of Gene Expression
0:50
Three Step Process
1:06
Illustration of Transcription Bubble
1:17
Transcription Starting Site is +1
5:15
Transcription Unit Extends From the Promoter to the Termination Region
5:40
Example 1
6:03
Eukaryotic Transcription: Initiation
14:27
RNA Polymerase II Binds to TATA Box to Initiate RNA Synthesis
14:34
TATA Binding Protein Binds the TATA Box
14:50
TBP Associated Factors Bind
15:01
General Transcription Factors
15:22
Initiation Complex
15:30
Example 2
15:44
Eukaryotic Transcription
17:59
Elongation
18:07
FACT (Protein Dimer)
18:24
Eukaryotic Transcription: Termination
19:36
Polyadenylation is Linked to Termination
19:42
Poly-A Signals Near the End of the pre-mRNA Recruit to Bind and Cleave mRNA
20:00
Mature mRNA
20:27
Dissociate from Template DNA Strand
21:13
Example 3
21:53
Eukaryotic Transcription
25:49
RNA Polymerase I Transcribes a Single Gene That Encodes a Long rRNA Precursor
26:14
RNA Polymerase III Synthesizes tRNA, 5S rRNA, and Other Small ncRNA
29:11
Prokaryotic Transcription
32:04
Only One Multi-Subunit RNA Polymerase
32:38
Transcription and Translation Occurs Simultaneously
33:41
Prokaryotic Transcription - Initiation
38:18
Initial Binding Site
38:33
Pribnox Box
38:42
Prokaryotic Transcription - Elongation
39:15
Unwind Helix and Expand Replication Bubble
39:19
Synthesizes DNA
39:35
Sigma 70 Subunit is Released
39:50
Elongation Continues Until a Termination Sequence is Reached
40:08
Termination - Prokaryotes
40:17
Example 4
40:30
Example 5
43:58
Post-Transcriptional Modifications
47:15
Can Post Transcribe your rRNA, tRNA, mRNA
47:28
One Thing In Common
47:38
RNA Processing
47:51
Ribosomal RNA
47:52
Transfer RNA
49:08
Messenger RNA
50:41
RNA Processing - Capping
52:09
When Does Capping Occur
52:20
First RNA Processing Event
52:30
RNA Processing - Splicing
53:00
Process of Removing Introns and Rejoining Exons
53:01
Form Small Nuclear Ribonucleoproteins
53:46
Example 6
57:48
Alternative Splicing
1:00:06
Regulatory Gene Expression Process
1:00:27
Example
1:00:42
Example 7
1:02:53
Example 8
1:09:36
RNA Editing
1:11:06
Guide RNAs
1:11:25
Deamination
1:11:52
Example 9
1:13:50
Translation

1h 15m 1s

Intro
0:00
Lesson Overview
0:06
Linking Transcription to Translation
0:39
Making RNA from DNA
0:40
Occurs in Nucleus
0:59
Process of Synthesizing a Polypeptide from an mRNA Transcript
1:09
Codon
1:43
Overview of Translation
4:54
Ribosome Binding to an mRNA Searching for a START Codon
5:02
Charged tRNAs will Base Pair to mRNA via the Anticodon and Codon
5:37
Amino Acids Transferred and Linked to Peptide Bond
6:08
Spent tRNAs are Released
6:31
Process Continues Until a STOP Codon is Reached
6:55
Ribosome and Ribosomal Subunits
7:55
What Are Ribosomes?
8:03
Prokaryotes
8:42
Eukaryotes
10:06
Aminoacyl Site, Peptidyl tRNA Site, Empty Site
10:51
Major Steps of Translation
11:35
Charing of tRNA
11:37
Initiation
12:48
Elongation
13:09
Termination
13:47
“Charging” of tRNA
14:35
Aminoacyl-tRNA Synthetase
14:36
Class I
16:40
Class II
16:52
Important About This Reaction: It Is Highly Specific
17:10
ATP Energy is Required
18:42
Translation Initiation - Prokaryotes
18:56
Initiation Factor 3 Binds at the E-Site
19:09
Initiation Factor 1 Binds at the A-Site
20:15
Initiation Factor 2 and GTP Binds IF1
20:50
30S Subunit Associates with mRNA
21:05
N-Formyl-met-tRNA
22:34
Complete 30S Initiation Complex
23:49
IF3 Released and 50S Subunit Binds
24:07
IF1 and IF2 Released Yielding a Complete 70S Initiation Complex
24:24
Deformylase Removes Formyl Group
24:45
Example 1
25:11
Translation Initiation - Eukaryotes
29:35
Small Subunit is Already Associated with the Initiation tRNA
29:47
Formation of 43S Pre-Initiation Complex
30:02
Circularization of mRNA by eIF4
31:05
48S Pre-Initiation Complex
35:47
Example 2
38:57
Translation - Elongation
44:00
Charging, Initiation, Elongation, Termination All Happens Once
44:14
Incoming Charged tRNA Binds the Complementary Codon
44:31
Peptide Bond Formation
45:06
Translocation Occurs
46:05
tRNA Released
46:51
Example 3
47:11
Translation - Termination
55:26
Release Factors Terminate Translation When Ribosomes Come to a Stop Codon
55:38
Release Factors Are Proteins, Not tRNAs, and Do Not Carry an Amino Acid
55:50
Class I Release Factors
55:16
Class II Release Factors
57:03
Example 4
57:40
Review of Translation
1:01:15
Consequences of Altering the Genetic Code
1:02:40
Silent Mutations
1:03:37
Missense Mutations
1:04:24
Nonsense Mutations
1:05:28
Genetic Code
1:06:40
Consequences of Altering the Genetic Code
1:07:43
Frameshift Mutations
1:07:55
Sequence Example
1:08:07
V. Gene Regulation
Gene Regulation in Prokaryotes

45m 40s

Intro
0:00
Lesson Overview
0:08
Gene Regulation
0:50
Transcriptional Regulation
1:01
Regulatory Proteins Control Gene Expression
1:18
Bacterial Operons-Lac
1:58
Operon
2:02
Lactose Operon in E. Coli
2:31
Example 1
3:33
Lac Operon Genes
7:19
LacZ
7:25
LacY
7:40
LacA
7:55
LacI
8:10
Example 2
8:58
Bacterial Operons-Trp
17:47
Purpose is to Produce Trptophan
17:58
Regulated at Initiation Step of Transcription
18:04
Five Genes
18:07
Derepressible
18:11
Example 3
18:32
Bacteriophage Lambda
28:11
Virus That Infects E. Coli
28:24
Temperate Lifecycle
28:33
Example 4
30:34
Regulation of Translation
39:42
Binding of RNA by Proteins Near the Ribosome- Binding Site of the RNA
39:53
Intramolecular Base Pairing of mRNA to Hide Ribosome Binding Site
40:14
Post-transcriptional Regulation of rRNA
40:35
Example 5
40:08
Gene Regulation in Eukaryotes

1h 6m 6s

Intro
0:00
Lesson Overview
0:06
Eukaryotic Transcriptional Regulations
0:18
Transcription Factors
0:25
Insulator Protein
0:55
Example 1
1:44
Locus Control Regions
4:00
Illustration
4:06
Long Range Regulatory Elements That Enhance Expressions of Linked Genes
5:40
Allows Order Transcription of Downstream Genes
6:07
(Ligand) Signal Transduction
8:12
Occurs When an Extracellular Signaling Molecule Activates a Specific Receptor Located on the Cell
8:19
Examples
9:10
N F Kappa B
10:01
Dimeric Protein That Controls Transcription
10:02
Ligands
10:29
Example 2
11:04
JAK/ STAT Pathway
13:19
Turned on by a Cytokine
13:23
What is JAK
13:34
What is STAT
13:58
Illustration
14:38
Example 3
17:00
Seven-Spanner Receptors
20:49
Illustration: What Is It
21:01
Ligand Binding That Is Activating a Process
21:46
How This Happens
22:17
Example 4
24:23
Nuclear Receptor Proteins (NRPs)
28:45
Sense Steroid and Thyroid Hormones
28:56
Steroid Hormones Bind Cytoplasmic NRP Homodimer
29:10
Hormone Binds NRP Heterodimers Already Present in the Nucleus
30:11
Unbound Heterodimeric NRPs Can Cause Deacetylation of Lysines of Histone Tails
30:54
RNA Interference
32:01
RNA Induced Silencing Complex (RISC)
32:39
RNAi
33:54
RISC Pathway
34:34
Activated RISC Complex
34:41
Process
34:55
Example
39:27
Translational Regulation
41:17
Global Regulation
41:37
Competitive Binding of 5 Prime CAP of mRNA
42:34
Translation-Dependent Regulation
44:56
Nonsense Mediated mRNA Decay
45:23
Nonstop Mediated mRNA Decay
46:17
Epigenetics
48:53
Inherited Patterns of Gene Expression Resulting from Chromatin Alteration
49:15
Three Ways to Happen
50:17
DNA Sequence Does Not Act Alone in Passing Genetic Information to Future Generations
50:30
DNA Methylation
50:57
Occurs at CpG Sites Via DNA Methyltransferase Enzymes
50:58
CpG Islands Are Regions with a High Frequency of CpG Sites
52:49
Methylation of Multiple CpG Sites Silence Nearby Gene Transcription
53:32
DNA Methylation
53:46
Pattern Can Be Passed to Daughter Cells
53:47
Prevents SP1 Transcription Factors From Binding to CpG Island
54:02
MECP2
54:10
Example 5
55:27
Nucleosomes
56:48
Histone Core
57:00
Histone Protein
57:03
Chromosome Condensation Via J1
57:32
Linker Histone H1
57:33
Compact DNA
57:37
Histone Code
57:54
Post-translational Modifications of N-Terminal Histone Tails is Part of the Epigenetic Code
57:55
Phosphorylation, Acetylation, Methylation, Ubiquitination
58:09
Example 6
58:52
Nucleosome Assembly
59:13
Duplication of DNA Requires Duplication of Histones by New Protein Synthesis
59:14
Old Histones are Recycled
59:24
Parental H3-H4 Tetramers
58:57
Example 7
1:00:05
Chromatin Remodeling
1:01:48
Example 8
1:02:36
Transcriptionally Repressed State
1:02:45
Acetylation of Histones
1:02:54
Polycomb Repressors
1:03:19
PRC2 Protein Complex
1:03:38
PRC1 Protein Complex
1:04:02
MLL Protein Complex
1:04:09
VI. Biotechnology and Applications to Medicine
Basic Molecular Biology Research Techniques

1h 8m 41s

Intro
0:00
Lesson Overview
0:10
Gel Electraophoresis
0:31
What is Gel Electraophoresis
0:33
Nucleic Acids
0:50
Gel Matrix
1:41
Topology
2:18
Example 1
2:50
Restriction Endonucleases
8:07
Produced by Bacteria
8:08
Sequence Specific DNA Binding Proteins
8:36
Blunt or Overhanging Sticky Ends
9:04
Length Determines Approximate Cleavage Frequency
10:30
Cloning
11:18
What is Cloning
11:29
How It Works
12:12
Ampicillin Example
12:55
Example 2
13:19
Creating a Genomic DNA Library
19:33
Library Prep
19:35
DNA is Cut to Appropriate Sizes and Ligated Into Vector
20:04
Cloning
20:11
Transform Bacteria
20:19
Total Collection Represents the Whole Genome
20:29
Polymerase Chain Reaction
20:54
Molecular Biology Technique to Amplify a Small Number of DNA Molecules to Millions of Copies
21:04
Automated Process Now
21:22
Taq Polymerase and Thermocycler
21:38
Molecular Requirements
22:32
Steps of PCR
23:40
Example 3
24:42
Example 4
34:45
Southern Blot
35:25
Detect DNA
35:44
How It Works
35:50
Western Blot
37:13
Detects Proteins of Interest
37:14
How It Works
37:20
Northern Blot
39:08
Detects an RNA Sequence of Interest
39:09
How It Works
39:21
Illustration Sample
40:12
Complementary DNA (cDNA) Synthesis
41:18
Complementary Synthesis
41:19
Isolate mRNA from Total RNA
41:59
Quantitative PCR (qPCR)
44:14
Technique for Quantifying the Amount of cDNA and mRNA Transcriptions
44:29
Measure of Gene Expression
44:56
Illustration of Read Out of qPCR Machine
45:23
Analysis of the Transcriptome-Micrarrays
46:15
Collection of All Transcripts in the Cell
46:16
Microarrays
46:35
Each Spot Represents a Gene
47:20
RNA Sequencing
49:25
DNA Sequencing
50:08
Sanger Sequencing
50:21
Dideoxynucleotides
50:31
Primer Annealed to a DNA Region of Interest
51:50
Additional Presence of a Small Proportion of a ddNTPs
52:18
Example
52:49
DNA Sequencing Gel
53:13
Four Different Reactions are Performed
53:26
Each Reaction is Run in a Lane of a Denaturing Polyacrylamide Gel
53:34
Example 5
53:54
High Throughput DNA Sequencing
57:51
Dideoxy Sequencing Reactions Are Carried Out in Large Batches
57:52
Sequencing Reactions are Carried Out All Together in a Single Reaction
58:26
Molecules Separated Based on Size
59:19
DNA Molecules Cross a Laser Light
59:30
Assembling the Sequences
1:00:38
Genomes is Sequenced with 5-10x Coverage
1:00:39
Compare Genomes
1:01:47
Entered Into Database and the Rest is Computational
1:02:02
Overlapping Sequences are Ordered Into Contiguous Sequences
1:02:17
Example 6
1:03:25
Example 7
1:05:27
VII. Ethics of Modern Science
Genome Editing, Synthetic Biology, & the Ethics of Modern Science

45m 6s

Intro
0:00
Lesson Overview
0:47
Genome Editing
1:37
What is Genome Editing
1:43
How It Works
2:03
Four Families of Engineered Nucleases in Use
2:25
Example 1
3:03
Gene Therapy
9:37
Delivery of Nucleic Acids Into a Patient’s Cells a Treatment for Disease
9:38
Timeline of Events
10:30
Example 2
11:03
Gene Therapy
12:37
Ethical Questions
12:38
Genetic Engineering
12:42
Gene Doping
13:10
Synthetic Biology
13:44
Design and Manufacture of Biological Components That Do Not Exist in Nature
13:53
First Synthetic Cell Example
14:12
Ethical Questions
16:16
Stem Cell Biology
18:01
Use Stem Cells to Treat or Prevent Diseases
18:12
Treatment Uses
19:56
Ethical Questions
20:33
Selected Topic of Ethical Debate
21:30
Research Ethics
28:02
Application of Fundamental Ethical Principles
28:07
Examples
28:20
Declaration of Helsinki
28:33
Basic Principles of the Declaration of Helsinki
28:57
Utmost Importance: Respect for the Patient
29:04
Researcher’s Duty is Solely to the Patient or Volunteer
29:32
Incompetent Research Participant
30:09
Right Vs Wrong
30:29
Ethics
32:40
Dolly the Sheep
32:46
Ethical Questions
33:59
Rational Reasoning and Justification
35:08
Example 3
35:17
Example 4
38:00
Questions to Ponder
39:35
How to Answer
40:52
Do Your Own Research
41:00
Difficult for People Outside the Scientific Community
41:42
Many People Disagree Because They Do Not Understand
42:32
Media Cannot Be Expected to Understand Published Scientific Data on Their Own
42:43
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Gene Regulation in Prokaryotes

    Medium, 5 examples, 5 practice questions

  • Regulation of the amount of RNA that is made from each DNA sequence can be controlled transcriptionally or translationally.
  • Bacteria often utilize a unit of DNA containing several genes all under the control of a single promoter, called an operon.
  • The lactose operon is inducible, while the tryptophan operon is derepressible.
  • A virus, Bacteriophage λ, can infect bacteria and either lyse the cell or integrate into the host genome by controlling transcription and translation.
  • Regulation of translation often occurs via binding of RNA by proteins near the ribosome-binding site of the RNA.

Gene Regulation in Prokaryotes

At what step does most gene regulation occur?
  • Elongation of transcription
  • Initiation of transcription
  • Translation
  • Termination of translation
The lactose operon is an example of what type of regulation?
  • Repressible
  • Derepressible
  • Inducible
  • Attenuation
The tryptophan operon is an example of what type of regulation?
  • Repressible
  • Derepressible
  • Inducible
  • Translational
Binding of RNA by proteins near the ribosome-binding site of the RNA is an example of regulation at which of the following steps:
  • Elongation of transcription
  • Initiation of transcription
  • Translation
  • Termination of translation
True/False: The lysogenic phase of the bacteriophage life cycle occurs when CI protein levels are high.
  • True
  • False

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.

Answer

Gene Regulation in Prokaryotes

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Lesson Overview 0:08
  • Gene Regulation 0:50
    • Transcriptional Regulation
    • Regulatory Proteins Control Gene Expression
  • Bacterial Operons-Lac 1:58
    • Operon
    • Lactose Operon in E. Coli
  • Example 1 3:33
  • Lac Operon Genes 7:19
    • LacZ
    • LacY
    • LacA
    • LacI
  • Example 2 8:58
  • Bacterial Operons-Trp 17:47
    • Purpose is to Produce Trptophan
    • Regulated at Initiation Step of Transcription
    • Five Genes
    • Derepressible
  • Example 3 18:32
  • Bacteriophage Lambda 28:11
    • Virus That Infects E. Coli
    • Temperate Lifecycle
  • Example 4 30:34
  • Regulation of Translation 39:42
    • Binding of RNA by Proteins Near the Ribosome- Binding Site of the RNA
    • Intramolecular Base Pairing of mRNA to Hide Ribosome Binding Site
    • Post-transcriptional Regulation of rRNA
  • Example 5 40:08

Transcription: Gene Regulation in Prokaryotes

Hello, and welcome back to www.educator.com.0000

Today, we are going to talk about gene regulation in prokaryotes.0003

As an overview, we will first talk about transcription and regulation.0009

And then, we will move into the regulation of translation.0014

We are going to focus mainly on transcription and regulation because that is most often where gene regulation occurs.0017

We will talk about bacterial operons.0023

We will focus on two of the most popular ones taught in many university classes which is the lactose operon and the tryptophan operon.0026

And then, I will talk to you about the virus bacteriophage λ.0035

We will talk about its lytic or lysogenic growth phases.0039

Finally, as I said, we will talk about the regulation or translation.0044

What is gene regulation?0051

It is the regulation of the amount of RNA that is made from each individual gene DNA sequence.0053

This is transcriptional regulation.0062

This is often tied in the amount of protein that is produced from that gene.0066

Usually, if you decrease the amount of mRNA made, you will decrease the overall amount of protein that can be made.0069

Regulatory proteins control our gene expression.0078

Most of that regulation occurs at the step of the initiation of transcription.0082

That is important.0088

Example, you could increase or decrease the binding ability of the RNA polymerase to its gene promoters.0094

Regulation can also occur during the elongation or termination steps on transcription, as well as during translation.0103

Let us jump right in, the bacterial operons.0120

What is an operon?0123

It is a functional unit of DNA that contains several genes, all under the control of a single promoter.0125

This is important, several genes all under the control of a single promoter.0135

That means all genes will be transcribed in equal quantities.0144

The first operon we are going to talk about, the lac operon or the lactose operon.0149

This is found in E. coli.0154

The whole purpose is to metabolize lactose.0156

This particular operon is regulated at the initiation step of transcription.0169

This is very common.0176

There are three genes contained in this operon, lacZ, lacY, and lacA.0178

It is what is called an enduciple system, meaning there is little or no transcription.0185

It can be induced to have high transcription.0197

When there is high lactose concentration, there will be high transcription.0200

If there is no lactose concentration, there will be no transcription.0206

Let us draw this out, what does it look like?0213

First of all, what does an operon look like?0217

We are not even specific, we are going to talk about the lactose one yet.0219

What is an operon look like, let us see.0222

If we check out our DNA, 5 prime, 3 prime.0225

We will say we have 4 genes here, the A gene and the B gene, the C gene and the D gene, all under a single promoter.0236

If we make the mRNA from that, we have what is called the polycistronic gene.0253

It is multiple genes on same mRNA.0263

We have our A, B, C, D genes.0267

What we have here is that, this is a fixed ratio of A to B to C to D because it is all under the control of the same gene promoter.0275

What this will make is then, for example, what this will make is protein A, protein B, protein C, and protein D.0294

Usually, what makes an operon is that all of the genes under the control of the single promoter, act together in a certain way.0337

They act in concert.0346

An example of that would be turning, let us say, we have substrate.0347

Let us go purple.0360

Protein A might work on substrate 1.0368

Protein A might be responsible for turning substrate 1 into substrate 2.0388

And then, protein B would be responsible for turning substrate 2 into 3, protein C, 3 into 4.0406

Protein D, 4 into 5.0414

What this entire operon does, what the entire operon does is turn substrate 1 into the final product of whatever product 5 is.0416

This is the ultimate goal of this entire operon.0429

It works in concert to do that.0434

Back to our specifics, let us look at the lac operon again.0438

We have the 3 genes, lacZ, that is going to encode an enzyme named β galactosidase.0442

That is the enzyme that cleaves lactose which is a disaccharide, into glucose and galactose, two monosaccharide.0451

We then have lacy which encodes lactose permeates,0460

that is a membrane protein responsible for allowing lactose to get into the cell.0465

It allows you to get from the outside to the inside.0473

And then, we have lacA encoding an enzyme called galactoside O acetyl transferase.0476

That is an enzyme involved in detoxification of the cell.0484

Also part of the lac operon is an upstream gene, it is not under the same promoter.0488

It is not being made in equal quantities of lacZ, Y, and A, in the normal thing.0495

LacI is our upstream gene that encodes a repressor protein, a transcription or repressor protein.0501

What this will do, it all bind lactose, if lactose is available.0508

Otherwise, it will bind the operator DNA sequence.0514

If this repressor binds the operator sequent, there is no transcription.0517

Basically, if lactose is available, the repressor is binding lactose and not the DNA operator.0525

Therefore, the operon is active.0533

Let us draw out this lac operon.0539

What is it look like, let us look at the DNA.0544

This is a way just to show that there is a lot of distance in the DNA.0571

It does not necessarily have to be very close.0575

This is the +1 site shown right here.0609

What we can make from that is our mRNA.0616

Our mRNA will have our lacZ, our lacY, and our lacA.0632

Right here, lacI, that is our repressor.0643

Cap, that is our cap binding sequence.0645

P is the promoter, always the operator.0649

Lac Z, Y, and A are our lac operon.0653

In times of high lactose, we will just write this out here.0659

We have high transcription.0671

In times of no lactose, we have no transcription.0676

For the promoter to be active, the cap protein must bind the cap DNA sequence.0689

For cap to bind, CAMP, cyclic AMP needs to be bound to the cap protein.0710

If there is no CAMP, then we have no cap DNA binding which means we have no transcription.0729

What is important is that, I will show you with the star.0761

As your glucose concentration increases, CAMP concentration decreases.0767

And that is going to lead to lower transcription.0798

Where does glucose come into play?0807

Remember, glucose is a breakdown product of lactose.0810

If I write this down here, lactose gets broken down by β galactosidase into glucose and galactose.0815

Glucose, in fact, blocks an enzyme called adenylyl cyclase.0843

That enzyme is responsible for turning ATP into CAMP.0864

As glucose concentration goes up, we block more of the enzyme that turned CAMP, that turns ATP into CAMP.0877

Therefore, as glucose concentration goes up, CAMP concentration goes down.0887

We see that CAMP needs to bind the cap protein, for the cap protein to bind the DNA sequence.0891

The cap protein is bind the DNA sequence, for transcription to occur.0902

It is all interrelated.0906

This is all important, we also know that lacI, the repressor protein that would be made,0910

normally binds the operator sequence right here, which will turn off transcription.0917

We do not get any mRNA, this is all blocked off, we would not do that.0923

If there is a bunch of lactose present, that lacI will actually bind the lactose,0932

therefore, freeing up the operator on so that transcription can occur.0939

There are couple caveats to this that I want to point out about the operator.0948

If the DNA sequence is mutated, then the repressor lacI cannot bind,0953

no matter what the lactose concentration in the cell is.0961

If the operator sequence is mutated and lacI cannot bind no matter what,0965

this is what would be called constitutively active or on, meaning high transcription.0970

That is if operator sequence is muted.0993

Another thing that can be altered is that, if the lacI gene is mutated,1007

then it does not matter how much lactose is present because the lacI gene would not be able to bind the operator sequence.1022

Therefore, we have the same result as up here, constitutively on, leading to high transcription.1037

These obviously are not under normal circumstances, that is when we have a mutation.1049

But this is just a couple other ways to see how this lac operon can be regulated or actually lose regulation.1057

Our second bacterial operon that we are going to talk about today is the tryptophan operon or the trp operon.1069

This is also found in E. coli, its purpose is to produce tryptophan from chrismic acid.1076

It is also regulated at the initiation step of transcription.1083

This one contains 5 genes instead of 3 and it is derepressible,1088

meaning that it is normally in a repressed state and you can take that repression off.1093

If you have very low tryptophan concentration around, this will become active because you are going to want to produce tryptophan.1100

Let us draw out the trp operon.1113

It has its own version of a repressor, that is upstream.1131

We have the promoter, we have the operator.1136

We have these reasons called 1, 2, 3, and 4 which are part of in attenuation region.1139

Then, we jump into the operon trp-E, trp-D, trp-C, trp-B, trp-A, obviously more DNA.1148

What does our mRNA look like?1170

The +1 site is right here.1174

We can get two different mRNAs.1178

This right here, remember, I said was called the attenuation region.1183

This will affect whether we get a small mRNA or a large mRNA.1190

And that will be due to the fact that, one thing I want to point out is that all of these are inverted repeats.1196

A thing about inverted repeat, they can bind to each other, causing intramolecular base pairing.1210

Another thing I want to point out is that sequence 1 contains 14 codons, 2 of which are tryptophan.1216

That is called the attenuation region.1238

First things first, we have our mRNA.1252

Here is our 5 prime, we have all the way through.1262

We have trp-B, trp-D, trp-C, trp-E, trp-A.1271

We will get this transcript, this is called an mRNA variant.1291

This one, when we have low tryptophan concentration.1296

When we have low tryptophan concentration, we get this.1307

The reason being, what happens is that we actually get this attenuation region looking like this.1310

We have 1, 2, 3, 4.1331

What this is, we call it a tennis racket or whatever you may want to call it.1342

We have intramolecular base pairing between region 2 and region 3.1347

Because we have low tryptophan, we are having this intramolecular base pairing between 2 and 3.1352

This leads to a ρ-dependent termination.1362

This mRNA is over 6,000 bases long and we know it is poly cystronic because it has all these genes.1374

There is the other variant that can be made when we have high tryptophan.1388

It will end here.1417

It is actually only 139 bases long.1419

This right here is the +139, right there is the +161 spot.1429

This will end up making a 34 hairpin.1442

Here I forgot to write out here.1455

What we have is EDCPA.1456

This is what we call the attenuated version.1481

This would undergo low independent termination.1484

This right here, this type of hairpin between the 3 and 4 actually causes the mRNA to pull out of the RNA polymerase.1494

It pulls it off the DNA resulting into an attenuated or shortened mRNA.1507

Whereas, the one up here, the hairpin between the 2 and 3 region is ρ-dependent.1512

It would not pull out until the ρ factor comes, after recognizing the stop sequence at the end of the mRNA.1518

That is the difference here.1526

When we have low trp, you have a 2 and 3 hairpin that allows you to transcribe all the way through to the end of the sequence.1527

We have high tryptophan concentration, you have a 3 -4 hairpin, up here in these attenuation regions,1538

which causes ρ-independent release of that mRNA.1545

In an attenuated, only 139 base mRNA.1549

One thing is important to mention is that, when we have this high tryptophan concentration,1556

the repressor will bind the operator sequence leading to no transcription.1565

Up here, when we have low tryptophan concentration,1576

the repressor does not bind the operator sequence which leads to transcription.1581

One thing to point out, remember, I said in this one sequence right here in the attenuation region,1594

it contains two tryptophan codons.1601

If you have absolutely 0 tryptophan, the ribosome will stall at that attenuation sequence 1 because it has 2 trp codons.1605

If you cannot put in trp right there, the ribosome cannot continue on synthesizing new protein.1634

This attenuation is a way to regulate gene transcription, about 8 to 10 fold.1644

You can do repression using that repressor protein gives you regulation about 70 fold.1654

Attenuation and regulation together can give you overall gene regulation, anywhere from around 600 folds.1659

These are just different ways between the lac operon and the trp operon of regulating gene expression.1667

In two completely different ways, even though they are both regulating at the same part of transcription which is the initiation.1674

Let us move away from our bacterial operons and let us switch gears to looking at how a virus works through its life cycle.1694

We have bacteriophage λ and that is a virus.1705

It infects the bacterium E. coli.1709

It has what is called a temperate lifecycle.1712

It can either be lysogenic which means its DNA genome.1714

Here is an example of a bacteriophage, it will latch onto an E. coli cell, this is E. coli with E. coli’s chromosome,1721

we will just say that.1736

The lambdaphage will shoot or inject its DNA into the E. coli.1739

It will make a circularized genome inside the E. coli.1748

This is the λ DNA.1753

There are two things that can be done.1761

Either this DNA can integrate into the host genome, becoming one with the E. coli DNA.1763

It can just stay there dormant or silent, kind of just outside of you.1773

Or the lambdaphage can just replicate its genome separately, over and over again.1778

Hijacking the E. coli cell machinery, making many new viruses.1789

It will just twice the bacterial cell and which in fact kills it and release more viruses to the environment.1795

In which case, those can do the same thing.1804

They can be infecting more E. coli.1806

Based on what they want to do, they can either integrate with the genome for lysogeny.1813

Or they can just hijack the cell machinery and use it to produce more viruses and lies the E. coli.1817

This is how the bacteriophage can affectively regulate its transcription to do one or the other.1825

Let us draw it out, it is a complex process.1835

I’m only going to draw out a simple part of it.1838

We are going to look here.1841

This would be the DNA of lamdaphage.1848

This is the λ DNA.1896

There is much more to it, we are just going to focus on this.1902

Right here, I’m just going to write it on then I will explain it.1908

Remember, this is DNA.1916

The cro protein than can be made from the cro DNA sequence will bind at this spot.1918

When cro binds here, this PL PRM PR PRE, those are all promoters and that is why they have their directionality.1937

It is which way they will promote transcription.1954

We have 3 of them pointing in the leftward direction.1957

Just PR pointing in the right direction.1959

If cro binds this region, this all of our 3 region, then that will block the PRM promoter.1961

This will lead to PR and PL, PR right here and PL.1977

They will be the promoters in charge of transcription.1991

This will lead to lysis of the bacterial cell.1996

C1 which is a protein that can be made from this DNA sequence, will bind to this sequence.2015

If C1 binds, the PR promoter is blocked.2034

And then, we will have PRN being the one that is transcribing.2045

This will lead to lysogeny, entering into the bacterial DNA.2058

Why would this occur?2075

This is important, I will write it with a star.2078

C1 concentration must be kept high to stay in lysogenic phase.2086

How it does that is, we have the pre-promoter helping out in this, helps keep C1 concentration high.2111

C1 is under control of this pre-promoter.2145

Basically, if the pre-promoter is active, C1 is produced quite highly.2150

If C1 is produced, we have it binding to this spot, blocking the PR promoter, which keeps this PRM promoter active.2157

It allows you to stay hidden inside the E. coli cell.2169

If you do not have C1 been made very high, we have cro being mixed.2175

If you are not going in the leftward direction, the leftward direction is producing C1, the rightward direction, the PRM PL.2180

The rightward is making cro.2197

If you are going rightward, we have a lot of cro around, cro protein.2201

That is going to bind this one, blocking the PRM.2204

The PRM and PRE are going to help with the C1.2208

Therefore, this is going to end up going through a lysis phase.2212

It is going to end up just hijacking the cell machinery, killing the bacteria and exiting out.2215

If you do not regulate transcription and translation properly, then you are not going to be able to contain,2222

whether you are going through lysogeny or lysis.2231

Other than transcriptional regulation, DNA damage can play into this.2234

For example, if we have bacterial DNA damage that can actually shift you from lysogeny to the lytic phase.2242

How does it do that?2268

When you have damage, you have DNA gaps occurring.2269

DNA gaps are bound by recA, binding the single stranded DNA.2279

RecA gets activated to recA*.2293

RecA* causes auto cleavage of the C1 protein which leads to lysis.2312

Not just transcription regulation, but actually DNA damage can affect2342

whether you have the lytic lifecycle or the lysogenic lifecycle of the bacteriophage.2347

This is just separate from an operon but it is a very involved DNA sequence that goes through transcription and regulation.2355

Depending on which protein is found in higher quantity, C1 or cro will tell you2364

whether you are going through lysogenic phase for C1 or lysis phase for cro protein.2372

I briefly want to go over the fact that we can be regulated at translation.2384

What that is normally looking like is that you bind the RNA, by proteins near the site that the ribosome will normally bind.2389

Therefore, the ribosome cannot even bind the mRNA.2399

This does not allow the 30S ribosomal subunit to bind RNA.2405

Another way that you can regulate translation is if you have intramolecular base pairing of mRNA.2411

The mRNA itself could be hiding its normal ribosome binding site.2416

Maybe it is right here, but because it is base paired to itself, the ribosome cannot bind the mRNA.2424

There is also post-transcriptional regulation of rRNA folding via translation repression of the ribosomal protein synthesis.2434

Basically, if you have rRNAs not folded correctly, they cannot interact properly2446

with the proteins to make the nuclear protein that we know as the ribosome.2453

I’m not going to go in any one of these specifically, but these are all options and possibilities how you can regulate translation.2459

For the last slide, I want to give you an example of something that is actually pretty cool phenomenon.2470

We have this thing, this piece of nucleic acid called the ssraRNA.2477

This is a really important RNA found in prokaryotes, that can rescue ribosomes2486

that are translating broken mRNAs or an mRNA galactose stop codon.2493

This is really important because if we have a ribosome and it is translating the mRNA, this is our eukaryote or prokaryotic.2500

This is the 50S and the 30S to make the 70S ribosome.2520

If we have an mRNA that is stuck in a ribosome, the broken mRNA, it gets stuck in the ribosome.2526

That ribosome will continue to try to translate it.2533

It is basically not happening.2538

Basically, what this does is it takes that ribosome out of commission and tell that mRNA can be released.2541

The ribosome is no longer useful for translating any functional proteins.2552

Same thing that if an mRNA does not have a stop codon, that can be problematic.2558

Our ribosomes need to be fully functional.2564

If we are translating broken mRNA, the ribosome is basically going to stall there and wait until something can be fixed.2568

Since that normally does not happen, we have this special RNA coming in and it will rescue that.2576

It is what is called a tmRNA.2581

It is part tRNA, part mRNA.2585

It can be charged with an alanine amino acid.2588

What happens is, if it is stalled right here, this tmRNA can come into the A site.2593

It can be charged with an alanine.2605

It can come into the A site, allow translocation to go to that P site.2613

The ribosome will be going that way which means that this will go to the P site.2624

What it does is translocation is able to occur.2631

And then, it is followed by 10 codons and a stop codon.2634

The protein is coming out, maybe it is stalled right there.2638

But then, you have this tag that is added.2645

It is 10 then a stop codon.2651

This is a very important piece.2659

This acts as a tag that directs the destruction of the mRNA by the cellular proteases.2663

That directs the overall protein that was produced, not the mRNA.2672

That protein gets it to the prodiuzome and gets destroyed.2683

This is really important, it allows the mRNA to be released from the ribosome.2686

Now, the ribosome can pick up on another mRNA and be functional, making a functional protein on that.2693

As well as, this messed up protein that was made from a broken mRNA or an mRNA without a stop codon,2698

can then be just degraded and thrown away.2706

That it does not fold into a protein and maybe become a protein that screws everything up.2709

Maybe has a function that was unintended.2716

This is a pretty cool RNA feature and a protective feature that is found for translational regulation in prokaryotes.2720

I hope that you enjoyed the lecture today.2732

Thank you for being here at www.educator.com and I hope to see you again.2735

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