1. Integrating Concepts in Biology
PowerPoint Slides for Chapter 2:
Central Dogma
2.2 How is gene transcription regulated?
by A. Malcolm Campbell, Laurie J. Heyer, & Christopher Paradise
Title Page
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

2. Biology Learning Objectives
Explain how genes are regulated to control RNA production
Demonstrate in writing and diagrams how proteins are made.
Bio-Math Learning Objectives
Interpret a graph of protein amounts to determine the strength of an inducer.
Use a position weight matrix to quantify how closely a sequence matches a previously observed pattern.
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

3. What tells RNA pol. when to make RNA?
Figure 2.7 Molecular structure of lactose. A, Lactose is a disaccharide, meaning it is made of two monosaccharides of (B) galactose and (C) glucose, which are connected by a beta chemical bond (pink arrow) connecting the circled atoms. Thick lines show atoms protruding toward the viewer, and dashed lines indicate atoms jutting away from the viewer.
Talking Point: Highlighting the two OH groups that will undergo condensation synthesis (H2O removed).
Fig. 2.7
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

4. Lactose Structure 1 two monosaccharides 2 Fig. 2.7
Figure 2.7 Molecular structure of lactose. A, Lactose is a disaccharide, meaning it is made of two monosaccharides of (B) galactose and (C) glucose, which are connected by a beta chemical bond (pink arrow) connecting the circled atoms. Thick lines show atoms protruding toward the viewer, and dashed lines indicate atoms jutting away from the viewer.
Talking Point: Highlighting the two OH groups that will undergo condensation synthesis (H2O removed). 2
Fig. 2.7
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

5. Lactose Structure 1 new covalent bond 2 one new disaccharide Fig. 2.7
Figure 2.7 Molecular structure of lactose. A, Lactose is a disaccharide, meaning it is made of two monosaccharides of (B) galactose and (C) glucose, which are connected by a beta chemical bond (pink arrow) connecting the circled atoms. Thick lines show atoms protruding toward the viewer, and dashed lines indicate atoms jutting away from the viewer.
Talking Point: One oxygen remains from the two OH groups. This disaccharide is cleaved into monosaccharides by the enzyme beta galactosidase. 2
one new disaccharide
Fig. 2.7
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

6. Lactose is Made and Degraded1
reversible reaction
Figure 2.7 Molecular structure of lactose. A, Lactose is a disaccharide, meaning it is made of two monosaccharides of (B) galactose and (C) glucose, which are connected by a beta chemical bond (pink arrow) connecting the circled atoms. Thick lines show atoms protruding toward the viewer, and dashed lines indicate atoms jutting away from the viewer.
Talking Point: One oxygen remains from the two OH groups. This disaccharide is cleaved into monosaccharides by the enzyme beta galactosidase. 2
Fig. 2.7
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

7. b-galactosidase Degrades Lactose1
b-galactosidase
Figure 2.7 Molecular structure of lactose. A, Lactose is a disaccharide, meaning it is made of two monosaccharides of (B) galactose and (C) glucose, which are connected by a beta chemical bond (pink arrow) connecting the circled atoms. Thick lines show atoms protruding toward the viewer, and dashed lines indicate atoms jutting away from the viewer.
Talking Point: One oxygen remains from the two OH groups. This disaccharide is cleaved into monosaccharides by the enzyme beta galactosidase. 2
Fig. 2.7
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

8. b-galactosidase Induction
Three phases
to this graph
Figure 2.8 Induction of beta-galactosidase after exposure to lactose. Lactose was added to growing E. coli cells and later removed. Aliquots of cells were isolated over time and tested for the amount of beta-galactosidase. The X-axis shows cell growth by increasing total protein mass as an indirect measure of time.
Talking Point: Highlighting the pre, post and during gene induction as revealed by protein production.
Fig. 2.8
modified from Jacob and Monod. 1961

9. b-galactosidase Induction
Figure 2.8 Induction of beta-galactosidase after exposure to lactose. Lactose was added to growing E. coli cells and later removed. Aliquots of cells were isolated over time and tested for the amount of beta-galactosidase. The X-axis shows cell growth by increasing total protein mass as an indirect measure of time.
Talking Point: First phase with no enzyme produced without lactose present.
phase 1:
pre-lactose
Fig. 2.8
modified from Jacob and Monod. 1961

10. b-galactosidase Induction
phase 2:
lactose present
Figure 2.8 Induction of beta-galactosidase after exposure to lactose. Lactose was added to growing E. coli cells and later removed. Aliquots of cells were isolated over time and tested for the amount of beta-galactosidase. The X-axis shows cell growth by increasing total protein mass as an indirect measure of time.
Talking Point: Rapid gene response to lactose and increase in the proportion of total protein that is beta-galactosidase.
Fig. 2.8
modified from Jacob and Monod. 1961

11. b-galactosidase Induction
phase 3:
lactose gone
Figure 2.8 Induction of beta-galactosidase after exposure to lactose. Lactose was added to growing E. coli cells and later removed. Aliquots of cells were isolated over time and tested for the amount of beta-galactosidase. The X-axis shows cell growth by increasing total protein mass as an indirect measure of time.
Talking Point: When lactose is removed, no more enzyme is produced by the cell, but previous enzyme persists for some time.
Fig. 2.8
modified from Jacob and Monod. 1961

12. b-galactosidase Induction
increased when
lactose present
Figure 2.8 Induction of beta-galactosidase after exposure to lactose. Lactose was added to growing E. coli cells and later removed. Aliquots of cells were isolated over time and tested for the amount of beta-galactosidase. The X-axis shows cell growth by increasing total protein mass as an indirect measure of time.
Talking Point: Conclusion point but need to emphasize increase in proportion of protein that is enzyme of interest.
Fig. 2.8
modified from Jacob and Monod. 1961

13. Possible Induction Mechanism
imports lactose
degrades
lactose
genes blocked
no lactose present
Figure 2.9 Proposed mechanism for activation of lactose-responsive genes. Jacob and Monod deduced that the two genes were (A) repressed by some common molecule (red circle/slash) and (B) activated by lactose. The red circle/slash represents a repressor protein.
Talking Point: The investigators hypothesized the existence of a protein that blocks transcription of the two genes.
Fig. 2.9
modified from Jacob and Monod. 1961

14. Possible Induction Mechanism
lactose present
block
removed
imports lactose
Figure 2.9 Proposed mechanism for activation of lactose-responsive genes. Jacob and Monod deduced that the two genes were (A) repressed by some common molecule (red circle/slash) and (B) activated by lactose. The red circle/slash represents a repressor protein.
Talking Point: Addition of the sugar lactose inactivates the two repressive proteins as if they were destroyed. The two genes are no longer repressed and can become activated.
lactose
degrades
lactose
Fig. 2.9
modified from Jacob and Monod. 1961

15. Genes Active Only When Needed
lactose absent
Figure 2.9 Proposed mechanism for activation of lactose-responsive genes. Jacob and Monod deduced that the two genes were (A) repressed by some common molecule (red circle/slash) and (B) activated by lactose. The red circle/slash represents a repressor protein.
Talking Point: Addition of the sugar lactose inactivates the two repressive proteins as if they were destroyed. The two genes are no longer repressed and can become activated.
lactose present
Fig. 2.9
modified from Jacob and Monod. 1961

16. Genetic Map of Lactose Digestion
all four genetic elements
in a single locus
Figure 2.10 Genetic map of DNA involved in lactose metabolism. The sizes of the boxes do not imply sizes of genes or the operator.
Talking Point: Slide points out that three protein-coding genes and one regulator are all near each other.
Fig. 2.10
modified from Jacob and Monod. 1961

17. Genetic Map of Lactose Digestion
operon = 1 promoter for >1 gene
Figure 2.10 Genetic map of DNA involved in lactose metabolism. The sizes of the boxes do not imply sizes of genes or the operator.
Talking Point: Slide points out that three protein-coding genes and one regulator are all near each other.
Fig. 2.10
modified from Jacob and Monod. 1961

18. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: Data showing the production of both proteins in the presence or absence of inducer lactose.
Table 2.1
modified from Jacob and Monod. 1961

19. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
haploids
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: First, note which cells are haploid.
Table 2.1
modified from Jacob and Monod. 1961

20. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
diploids
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: And which ones are diploid using this common genetic nomenclature.
Table 2.1
modified from Jacob and Monod. 1961

21. Table 2.1 no lactose lactose Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
no lactose
lactose
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: Highlighting the columns when lactose was absent but glucose was present.
Table 2.1
modified from Jacob and Monod. 1961

22. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: Wild-type haploid cell produces very little beta-galactosidase in the absence of lactose but a lot (defined as 100% for comparison sake) when lactose added.
Table 2.1
modified from Jacob and Monod. 1961

23. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: When the inhibitor gene was deleted, the beta-galactosidase gene was always activated.
Table 2.1
modified from Jacob and Monod. 1961

24. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: Diploid cells produced more than twice as much beta-galactosidase when lactose was present and glucose absent.
Table 2.1
modified from Jacob and Monod. 1961

25. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: When a dominant allele of the inhibitor was used in a haploid, lactose was unable to induce the beta-galactosidase gene.
Table 2.1
modified from Jacob and Monod. 1961

26. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: In diploid cell, the dominant inhibitor allele was able to produce enough inhibitor protein to shut down both beta-galactosidase alleles.
Table 2.1
modified from Jacob and Monod. 1961

27. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: The operator does not code for any protein but when it is deleted, beta-galactosidase is not able to be produced even when lactose is present and glucose absent. The operator needs to be functional to activate the gene it regulates.
Table 2.1l
modified from Jacob and Monod. 1961

28. Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: A critical experiment that shows operators only work on the allele attached to them and unlike inhibitors cannot affect the distant allele so beta-galactosidase is produced at haploid levels of protein even though the cell is diploid for the galactosidase gene.
Table 2.1
modified from Jacob and Monod. 1961

29. Table 2.1 same for permease Table 2.1
Table 2.1 Percent synthesis of b-galactosidase in diploid and haploid E. coli.
Table 2.1 Percent synthesis of beta-galactosidase in diploid and haploid E. coli.
Talking Point: The beta-galactosidase and permease results are nearly identical except the loss of inhibitor produces less permease when the hapliod cell lacks inihibtior. This is consistent with the permease gene being further away from the operator.
same for permease
Table 2.1
modified from Jacob and Monod. 1961

30. Regulation of Lac Genes
Figure 2.11 Schematic diagrams of lac gene regulation. A, lacI+ encodes an inhibitor that binds to lacO+ and blocks transcription. B, In the presence of lactose, LacI+ protein is unable to inhibit lacO+ and transcription of lacB and lacP genes.
Talking Point: Improved model showing the inhibitor blocks the ability of the operator to activate the downstream genes. Lactose inhibits the inhibitor.
Fig. 2.11
modified from Jacob and Monod. 1961

31. Genetic Circuit Diagrams
Figure 2.11 Schematic diagrams of lac gene regulation. A, lacI+ encodes an inhibitor that binds to lacO+ and blocks transcription. B, In the presence of lactose, LacI+ protein is unable to inhibit lacO+ and transcription of lacB and lacP genes.
Talking Point: Improved model showing the inhibitor blocks the ability of the operator to activate the downstream genes. Lactose inhibits the inhibitor.
Fig. 2.11
modified from Jacob and Monod. 1961

32. Regulation of Lac Genes
2 genes blocked
no lactose present
Figure 2.11 Schematic diagrams of lac gene regulation. A, lacI+ encodes an inhibitor that binds to lacO+ and blocks transcription. B, In the presence of lactose, LacI+ protein is unable to inhibit lacO+ and transcription of lacB and lacP genes.
Talking Point: Absence of lactose or the presence of glucose blocks the activation of the two genes by binding to the operator.
Fig. 2.11
modified from Jacob and Monod. 1961

33. Regulation of Lac Genes
lactose present
block removed
Figure 2.11 Schematic diagrams of lac gene regulation. A, lacI+ encodes an inhibitor that binds to lacO+ and blocks transcription. B, In the presence of lactose, LacI+ protein is unable to inhibit lacO+ and transcription of lacB and lacP genes.
Talking Point: Removal of the protein inhibitor by the sugar lactose (and no glucose) removes blockage of the operator and the two genes can be transcribed by RNA polymerase to produce mRNA.
2 genes on
Fig. 2.11
modified from Jacob and Monod. 1961

34. LacI+ Bound to lacO+ LacI protein Figure 2.11c
Figure 2.11c Schematic diagrams of lac gene regulation. Figure 2.11 Schematic diagrams of lac gene regulation. C, Molecular structure of LacI+ bound to lacO+. Four molecules of LacI+ protein (green) coordinate their shapes to bind to two specific DNA sites within lacO+ (orange DNA).
Talking point: Slide points out the two colored, four protein LacI protein complex.
LacI protein
Figure 2.11c
courtesy David Goodsell

35. LacI+ Bound to lacO+ lacO+ Figure 2.11c
Figure 2.11c Schematic diagrams of lac gene regulation. Figure 2.11 Schematic diagrams of lac gene regulation. C, Molecular structure of LacI+ bound to lacO+. Four molecules of LacI+ protein (green) coordinate their shapes to bind to two specific DNA sites within lacO+ (orange DNA).
Talking point: The operator is the portion bent into the loop of DNA.
Figure 2.11c
courtesy David Goodsell

36. LacI+ Bound to lacO+ lacO+ RNA polymerase blocked Figure 2.11c
Figure 2.11c Schematic diagrams of lac gene regulation. Figure 2.11 Schematic diagrams of lac gene regulation. C, Molecular structure of LacI+ bound to lacO+. Four molecules of LacI+ protein (green) coordinate their shapes to bind to two specific DNA sites within lacO+ (orange DNA).
Talking point: The operator is the portion bent into the loop of DNA.
RNA polymerase blocked
Figure 2.11c
courtesy David Goodsell

37. T7 Promoter Bound with RNA pol
T7 promoter region
Figure 2.13 Sequences of the T7 promoter and mRNA. Both strands of the protected T7 DNA segment after DNase treatment are in red. The mRNA sequence of the T7 gene used for the experiment is in blue. Underlined portions highlight sequence similarity.
Talking Point: Note that the top strand of the DNA matches the mRNA sequence.
Fig. 2.12
modified from Pribnow

38. T7 Promoter and mRNA start codon Fig. 2.12
Figure 2.13 Sequences of the T7 promoter and mRNA. Both strands of the protected T7 DNA segment after DNase treatment are in red. The mRNA sequence of the T7 gene used for the experiment is in blue. Underlined portions highlight sequence similarity.
Talking Point: Start codon is further downstream (to the right).
Fig. 2.12
modified from Pribnow

39. RNA Polymerase Binding to DNA
Figure 2.13 Sequences of the T7 promoter and mRNA. Both strands of the protected T7 DNA segment after DNase treatment are in red. The mRNA sequence of the T7 gene used for the experiment is in blue. Underlined portions highlight sequence similarity.
Talking Point: RNA plolymerase binds to promoter and the first few bases that will be transcribed upstream of the start codon.
Fig. 2.12
modified from Pribnow

40. Bacterial Promoter Alignments
start transcription
Figure 2.13 Alignment of DNA sequences containing promoters. Sequences from viral (V) and bacterial (B) genes are aligned so that conserved bases are in the boxed region and the first transcribed bases (underlined) are nearby.
Talking Point: Start transcription site is boxed in green. TATA-region boxed in red.
Fig. 2.13
modified from Pribnow

41. Bacterial Promoter Alignments
“conserved”
bases and
location
Figure 2.13 Alignment of DNA sequences containing promoters. Sequences from viral (V) and bacterial (B) genes are aligned so that conserved bases are in the boxed region and the first transcribed bases (underlined) are nearby.
Talking Point: Conserved bases of promoter are not universally conserved. How do we quantify conservation? Bioinformatics is required.
Fig. 2.13
modified from Pribnow

42. Used with IQ 19 human interferon g gene mRNA Unmarked Figure 2.2
Unmarked Figure 2.2 to go with Integrating Question 19.
Talking Point: mRNA sequence highlighted in red.
mRNA
Unmarked Figure 2.2
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

43. Used with IQ 19 “conserved” bases Unmarked Figure 2.2
Unmarked Figure 2.2 to go with Integrating Question 21.
Talking Point: Loosely conserved TATA-rich area of promoter.
Unmarked Figure 2.2
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

44. BioMath Exploration 2.2 step 1: frequency of each base BME 2.2 Tables
Step 1: Make a table of the proportion of A, C, G and T in each position of the boxed pattern within the known promoters. For example, the first nucleotide inside the box in Figure 2.13 is a T in five of the six sequences (83%) and a G in one of the six sequences (17%). The results of this step are shown in Table BME 2.1.
Talking Point: Fist step in position weight matrix is to find the frequency of each base for each position.
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

45. BioMath Exploration 2.2 BME 2.2 Tables
Step 1: Make a table of the proportion of A, C, G and T in each position of the pattern within the box. For example, the first nucleotide inside the box in Figure 2.14 is a T in five of the six sequences (83%) and a G in one of the six sequences (17%).
Talking Point: Connecting the 6 known promoters with the first step in the process of calculating the PWM.
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

46. BioMath Exploration 2.2 BME 2.2 Tables
Step 1: Make a table of the proportion of A, C, G and T in each position of the pattern within the box. For example, the first nucleotide inside the box in Figure 2.14 is a T in five of the six sequences (83%) and a G in one of the six sequences (17%).
Talking Point: Connecting the 6 known promoters with the first step in the process of calculating the PWM.
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

47. BioMath Exploration 2.2 step 2: divide A & T #s by 0.28
Step 2: Make a new table (Table BME 2.2) by dividing each entry in the A row of Table BME 2.1 by the proportion of As (0.28) in the entire genome, and similarly for the C (0.22), G (0.22), and T (0.28) rows with the following exception: Everywhere there is a 0 in Table BME 2.1, put a 0.01 in the corresponding spot in the new table.
Talking Point: Step 2 divides the frequency observed by the overall genome frequency of each base.
step 2:
divide A & T #s by 0.28
divide G & C #s by 0.22
convert 0s to 0.01
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

48. BioMath Exploration 2.2 step 3: log2 transform all #s BME 2.2 Tables
Step 2: Make a new table by dividing each entry in the A row from Step 1 by the proportion of A’s in the entire genome, and similarly for the C, G, and T rows, with the following exception: everywhere there is a 0 in the table from Step 1, put a 0.01 in the new table. In this example, the 0 in position 1 of the A row above means that 0.01 is in the corresponding position of the table below. Assume that the proportion of A’s and T’s in the genome is 0.28 each, and the proportion of C’s and G’s is 0.22 each: Talking Point: Convert these numbers to log_2 values. This is why zeros were converted to 0.01 values in step 2.
step 3: log2 transform all #s
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

49. BioMath Exploration 2.2 position weight matrix BME 2.2 Tables
Step 3: Compute the base 2 logarithm of each number in Table BME 2.2. {Connections: Base 2 logarithms were described in Bio-Math Exploration 1.3.}
Talking Point: Final PWM allows you to determine whether a sequence is likely to be a promoter in the species under consideration. Values above 1 are likely real promoters while negative numbers indicate low probability of promoter.
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

50. TATGATG BioMath Exploration 2.2 position weight matrix BME 2.2 Tables
Step 3: Compute the base 2 logarithm of each number in the table from Step 2. The resulting table is called a position weight matrix.
Talking Point: The sequence in green is probably a promoter since all if its values are optimal as determined by the consensus PWM.
BME 2.2 Tables
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

51. Integrating Question #17
TATGATG
Go to this WebLogo website ( and submit the six rows of bases from the boxed alignment in Figure 2.13.
Talking Point: Students can use bioinformatics tools to generate a visual position weight matrix in this sequence logo.
web logo from Figure 2.13
Integrating Question #17
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

52. ssDNA Promoter Sequence
Figure 2.14 Testing sequence specificity of promoters. A, Single-stranded DNA sequence from a chicken gene promoter. The negative numbers indicate the number of bases between that site and the beginning of transcription.
Talking Point: Start transcription is 25 base pairs to the right of the A furthest to the right.
Fig. 2.14a
modified from Wasylyk and Chambon. 1981

53. Functional Testing of Promoters
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Experimental testing of whether sequence is important for a promoter’s function, or just AT-rich DNA.
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

54. Functional Testing of Promoters
RNA size markers in bases
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Start analysis with MW markers for orientation.
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

55. Functional Testing of Promoters
mRNAs produced from 3
different promoters
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Design included one positive control (wt) and two experimental sequences.
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

56. Functional Testing of Promoters
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Highlighting the two subtle changes in potential promoters.
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

57. Functional Testing of Promoters- +
mRNA
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Begin with results from control condition. mRNA produced only when RNA polymerase was added to potential promoter and gene.
3 - RNA pol
4 + RNA pol
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

58. Functional Testing of Promoters- + - +
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: TAGA contains a G and thus “melts” less easily and was predicted to function poorly as a promoter.
1 - RNA pol
2 + RNA pol
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

59. Functional Testing of Promoters- + - + - +
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: TAAA was predicted to function as well as TATA wt, but it did not. TAGA did better.
5 - RNA pol
6 + RNA pol
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

60. Functional Testing of Promoters
TAGA > TAAA
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Conclusion of promoter strengths.
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

61. Functional Testing of Promoters- + - + - +
Figure 2.14 Testing sequence specificity of promoters. B, Electrophoresis results from mRNA (open arrowheads) produced using three variations of promoters with the wild-type (wt) sequence shown in A. The amount of darkness indicates the quantity of mRNA transcribed. The promoter sequences are listed at the top of the lanes; even lanes contained RNA polymerase, and odd lanes did not.
Talking Point: Conclusion is that AT-rich is insufficient to contain the promoter information. Sequence matters more than just AT-rich content.
promoters sequence-specific not just AT-rich
Fig. 2.14B
modified from Wasylyk and Chambon. 1981

62. Band Shift Analysis of Promoter
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Experimental testing of what proteins bind to the promoter.
Fig. 2.15
modified from
Killeen et al., 1992.

63. Band Shift Analysis of Promoter
do these proteins bind to a particular promoter?
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Highlighting organization of experimental design. Overall question is posed.
Fig. 2.15
modified from
Killeen et al., 1992.

64. Band Shift Analysis of Promoter
Ingredients:
3 transcription factors (TF)
RNA polymerase
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: 4 proteins tested for their ability to bind to a promoter sequence. Promoter DNA is the only radioactive component and X-ray film is used to detect radiation from promoter. X-rays are NOT used in this experiment (point of confusion for many students)
Radioactive promoter (DNA)
Fig. 2.15
modified from
Killeen et al., 1992.

65. Band Shift Analysis of Promoter
method: gel electrophoresis (non-denaturing)
radioactive promoter
X-ray film exposure by radioactive DNA
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Summary of experimental method.
Fig. 2.15
modified from
Killeen et al., 1992.

66. Band Shift Analysis of Promoter
no proteins added to promoter
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Start with negative control lane on gel. Promoter is small as expected and no shift detected.
Fig. 2.15
modified from
Killeen et al., 1992.

67. Band Shift Analysis of Promoter
all proteins added to promoter
promoter + proteins =
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Positive control shows big shift in size for the promoter because all 4 proteins are bound.
Fig. 2.15
excess promoter
modified from
Killeen et al., 1992.

68. Band Shift Analysis of Promoter
2 TFs + RNA pol
<1%?
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Leaving out the TBP inhibits most of the band shift.
Fig. 2.15
modified from
Killeen et al., 1992.

69. Band Shift Analysis of Promoter
2 TFs + RNA pol
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Leaving out the transcription factor IIB inhibits all of the band shift.
Fig. 2.15
modified from
Killeen et al., 1992.

70. Band Shift Analysis of Promoter
2 TFs + RNA pol
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Leaving out the transcription factor RAP74 inhibits all of the band shift.
Fig. 2.15
modified from
Killeen et al., 1992.

71. Band Shift Analysis of Promoter
3 TFs - RNA pol
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Leaving out the RNA polymerase inhibits all of the band shift.
Fig. 2.15
modified from
Killeen et al., 1992.

72. Band Shift Analysis of Promoter
all 4 must be present to bind
Figure 2.15 X-ray film shows the results of a promoter-binding experiment. A radioactive DNA promoter sequence was incubated under six different conditions; combinations contained three transcription factors and RNA polymerase (RNA pol). Samples were loaded on the gel and separated by electrophoresis; promoter alone was loaded in the far left lane as a negative control. Plus signs indicate which components were added to the radioactive promoter.
Talking Point: Summary conclusion for experiment. Cooperative binding rather than piece-meal binding.
Fig. 2.15
modified from
Killeen et al., 1992.

73. Deletion Mapping of Promoter
manipulate promoter
measure phenotypes
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: Experimental testing of which part of a promoter enhances transcription of downstream gene.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

74. Deletion Mapping of Promoter
manipulate promoter
measure phenotypes
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: Experimental testing of which part of a promoter enhances transcription of downstream gene.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

75. Deletion Mapping of Promoter
fast
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: Experimental testing of which part of a promoter enhances transcription of downstream gene.
wild-type, + control
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

76. Deletion Mapping of Promoter
remove 65 bp
fast
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

77. Deletion Mapping of Promoter
remove 42 bp
medium
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

78. Deletion Mapping of Promoter
medium
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
promoter function
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

79. Deletion Mapping of Promoter
remove 35 bp
medium
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

80. Deletion Mapping of Promoter
remove 35 bp
none!
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

81. Deletion Mapping of Promoter
none!
grows?
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
promoter function
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

82. Deletion Mapping of Promoter
Figure 2.16 Mapping a promoter. Four experimental versions of a 320 base pair (bp) promoter placed upstream of an essential gene (orange arrow). Cells containing different promoters were measure for growth rate and resistance to a drug that could stop cell growth with low transcription.
Talking Point: 65 base pairs have not measureable function.
promoter has 2
functional regions
Fig. 2.16
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

83. Steroid Hormones progesterone testosterone a) b) Fig. 2.17
Figure 2.17 Steroid hormones. A, Progesterone and (B) testosterone can stimulate transcripts of particular genes. Carbons are present at vertices and the ends of line segments if no other letter is present.
Talking Points: Two steroid hormones. Both are hydrophobic.
Fig. 2.17
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

84. Characterizing Steroid Receptor
What kind of molecule is the receptor?
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: Experiment to determine what type of molecule functioned as the steroid (progesterone) receptor.
Fig. 2.18
modified from O’Malley et al., 1970.

85. Characterizing Steroid Receptor
100% progesterone binding
negative control
defines maximum
binding
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: This condition establishes maximum binding (100%).
Fig. 2.18
modified from O’Malley et al., 1970.

86. Characterizing Steroid Receptor
digest proteins….
80% less binding
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: Digesting proteins substantially reduced ability of steroid to bind to its receptor.
Fig. 2.18
modified from O’Malley et al., 1970.

87. Characterizing Steroid Receptor
digest RNA….
no change
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: Digesting RNA did not reduced ability of steroid to bind to its receptor.
Fig. 2.18
modified from O’Malley et al., 1970.

88. Characterizing Steroid Receptor
digest DNA….
slightly
less binding
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: Digesting DNA did not substantially reduced ability of steroid to bind to its receptor.
Fig. 2.18
modified from O’Malley et al., 1970.

89. Characterizing Steroid Receptor
digest DNA….
significant?
no error bars
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: Without error bars in the original data, it is impossible to determine if this reduction is significant. Probably not, so don’t dwell on this with students.
Fig. 2.18
modified from O’Malley et al., 1970.

90. Characterizing Steroid Receptor
likely
a protein
Figure 2.18 Amount of steroid bound by its receptor. Nuclei containing the injected steroid were incubated for 30 minutes with either pronase, RNase, or DNase, or no enzyme as a positive control. The amount of steroid bound to its receptor was quantified for each enzyme treatment, as well as the positive control.
Talking Point: The steroid receptor is protein. Steroid receptors are proteins for all other steroids tested.
Fig. 2.18
modified from O’Malley et al., 1970.

91. Localization of Steroid Receptor
two graphs in one space
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Cytoplasmic axis on the left and nuclear on the right.
over the same time period
Fig. 2.19
modified from O’Malley et al., 1970.

92. Localization of Steroid Receptor
cytoplasm decreases…..
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Gradual reduction of cytoplasmic receptor bound to radioactive steroid hormone.
Fig. 2.19
modified from O’Malley et al., 1970.

93. Localization of Steroid Receptor
nucleus increases…..
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Gradual increase of nuclear receptor bound to radioactive steroid hormone.
Fig. 2.19
modified from O’Malley et al., 1970.

94. Localization of Steroid Receptor
moves from cytoplasm….
….to nucleus.
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
Fig. 2.19
modified from O’Malley et al., 1970.

95. No Delay in Protein Binding
same protein,
relocated
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
Fig. 2.19
modified from O’Malley et al., 1970.

96. No Delay in Protein Binding
steroid
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
Fig. 2.19
modified from O’Malley et al., 1970.
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

97. No Delay in Protein Binding
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
Fig. 2.19
modified from O’Malley et al., 1970.
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

98. No Delay in Protein Binding
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
nucleus
Fig. 2.19
modified from O’Malley et al., 1970.
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved. 

99. No Delay in Protein Binding
Figure 2.19 Tracing progesterone location over time. Hens injected with radioactive progesterone for the indicated time when cells were disrupted. Cytoplasmic and nuclear progesterone-receptor complexes were quantified (µg/mL) at each time point.
Talking Point: Reciprocal nature of the two receptors with no time lag between them indicates the receptor never lets go of the hormone but changes is shape and location over time.
Fig. 2.19
modified from O’Malley et al., 1970.
Copyright © 2015 by AM Campbell, LJ Heyer, CJ Paradise. All rights reserved.