1. Regulation of Gene Expression
Chapter 11
Regulation of Gene Expression

2. 11.1 Several Strategies Are Used to Regulate Gene Expression
Gene expression is precisely regulated
Constitutive genes-are actively expressed all the time
Inducible genes-are expressed only when their proteins are needed by the cell
We will focus on inducible genes

3. Genes are subject to positive and negative regulation
DNA can be regulated at every step from genes to proteins
During transcription there is an important form of gene regulation
Genes that get transcribed are said to be active

4. Differential gene expression: expression of different genes in different kinds of cells, despite having the same genome

5. Two types of transcription factors (proteins) control whether or not a gene is active
Repressors
Activators
These bind to specific DNA sequences at or near the promoter and decide which genes to be transcribed

6. In negative regulation, a repressor binds near the promoter to prevent transcription
In positive regulation, the binding of an activator stimulates transcription

8. 11.2 Many prokaryotic genes are regulated in Operons
Prokaryotes conserve E by making proteins only when needed
The most efficient means of regulating gene expression is at the level of transcription

9. Regulating gene transcription
Inducible genes expression is switched on by an inducer
Example: prokaryotic bacteria do not create lactase if allactose (lactose) is not present. When allactose (inducer) is present the bacterial cell begins creating the protein lactase to metabolize the lactose (done with three proteins)

10. Lac Operon

11. Operons are units of transcriptional regulation in prokaryotes
Structural genes-a gene that encodes the primary structure of a protein not involved in the regulation of gene expression (example three genes that metabolize lactose)
A cluster of genes with a single promoter is called an operon and the operon that codes for the three-lactose metabolizing enzymes is called the lac operon

12. Lac Operon

13. The repressor proteins can bind very tightly with the operator
The lac operon had another DNA sequence called an operator, which is near the promoter and controls transcription of the structural genes
The repressor proteins can bind very tightly with the operator
An inducible operon is turned off unless needed
A repressible operon is turned on unless not needed

14. Lac Operon

15. Operator-repressor interactions regulate transcription in the lac and trp operons
In inducible lac operons, a repressor protein prevents transcription until the lac-encoded proteins are needed
The trp operon is a repressible operon that is turned off by a repressor only under some circumstances

16. Lac Operon
The lac operon is not transcribed unless lactose is the main sugar present
A repressor protein is normally bound to the operator, preventing transcription
When lactose is present it detaches from the operator which allows RNA polymerase to bind to the promoter and transcribe the structural genes

17. http://www. google. com/imgres

18. The key to this system is the repressor protein
The repressor is always present in the cell to occupy the operator and keep the operon turned off
Has a sport for the inducer to bind and when it binds allosteric regulation happens

19. Repressor

20. Lac operon animation

21. trp Operon
A repressible operon is switched off when its repressor is bound to its operator
In this case the repressor binds to the DNA only if a co-repressor is present
Co-repressor-a molecule that binds to the repressor which causes it to bind to the operator and inhibit transcription

22. trp operon

23. The trp operon’s structural genes catalyze the synthesis of the amino acid tryptophan and is a repressible operon
In high concentrations the cell wants to stop making the enzymes to create trp
Trp functions as a co-repressor that binds to the repressor of the trp operon and prevent transcription

24. trp operon

25. Summarize the differences between operons
Inducible: the inducer interacts with the repressor which causes it to not bind to the operator and halts transcription
Repressible: the product of the pathway which is the co-repressor binds to the repressor which then binds to the operator and halts transcription

26. Lac Operon

27. trp operon

28. RNA polymerase can be directed to a class of promoters
Sigma factors-proteins in prokaryotes that can bind to RNA polymerase and direct the polymerase to specific promoters
Example: when nutrients run out for bacteria the cell goes into a sporulation lifestyle and is dormant for a while. These genes are only created when directed by sigma factors

29. trp Operon

30. 11.3 Eukaryotic Genes Are Regulated by Transcription Factors and DNA Changes

31. Transcription factors act at eukaryotic promoters
A eukaryotic promoter is a region of DNA where RNA polymerase binds
The most common promoter is the TATA box because it has a lot of A-T base pairs
RNA P cannot bind to the promoter unless it has general transcription factors bind to the promoter first

32. General Transcription Factors

33. First, the protein TFIID binds to the TATA box which changes the shape of the DNA so other TF’s can bind
The RNA P then binds after other TF’s have bound to the DNA
These TF’s may be positive regulators (activators) or negative regulators (repressors) of transcription

34. General Transcription Factors

35. DNA sequences that binds activators are called enhancers, those that bind repressors are called silencers
When these bind to DNA sequences they interact with the RNA P complex and cause the DNA to bend
The combination of factors present determines the initiation of transcription
(2,000 different TF’s in humans so many different regulation possibilities)

37. Initiation of transcription
Example of Activation

38. Example in book lets read… (pg 217)
The expression of sets of genes can be coordinately regulated by transcription factors
Eukaryotes also use sigma factors to guide the RNA P to specific promoters
The expression of genes can be coordinated if they share regulatory sequences that bind the same transcription factors
Example in book lets read… (pg 217)

39. Epigenetic changes to DNA and chromatin can regulate transcription
Euk- can also regulate the transcription of large stretches of DNA
Do this by altering the DNA or the chromosomal proteins that package the DNA (this is reversible unlike mutations)
These are called epigenetic changes and can be passed on to daughter cells after mitosis or meiosis

40. Proteins that package the DNA

41. DNA methylation
Cytosine residues in the DNA are chemically modified by the addition of a methyl group (-CH3) to make 5-methyl cytosine
This is done by the enzyme DNA methyltransferase
Usually done to C’s that are adjacent to G’s
Areas that have a lot of the are called CpG Islands

43. This change is heritable and reversible
The methyl group can be removed by an enzyme named demethylase
Methylated DNA binds proteins that repress transcription so the gene may be silenced
Sometimes large stretches or whole chromosome can be Methylated
Euchromatin is transcribed and heterochromatin is Methylated and not transcribed

44. DNA methylation: represses transcription by condensing DNA
--For example, Barr bodies (inactivated X chromosomes in females) often have methylated DNA

45. Female bar body example… lets read page 219

46. Histone Protein Modification
Another mechanism for epigenetic gene regulation is the alteration of chromatin structure or chromatin remodeling
DNA is packaged as a nucleosome which is a core of positively charged histone proteins that DNA is wound around
Nucleosomes make it so RNA P cannot bind

48. Each histone has a tail of amino acids that sticks out of the structure
Enzymes called histone acetyltransferases add acetyl groups to these positively charged amino acids which neutralizes their charges
Usually there is a strong attraction between the proteins and DNA which is negatively charged
This then loosens the nucleosome and allows for transcription

49. Histone modifications: acetylation promotes transcription due to neutralization of positive charges so they don’t bind to neighboring nucleosomes (I remember “acetylation= activation”)

50. Acetyltransferases can activate transcription
Another chromatin remodeling protein histone deacetylase removes acetyl groups from histones and represses transcription
Histone protein modification video

51. Epigenetic changes can be induced by the environment
Epigenetic changes can be passed on to the next generation if done in a germline cell
Epigenetic genomes have been proven to be different by twin studies
Stress can be a factor for methylation

52. 11.4 Eukaryotic Gene Expression Can be Regulated after Transcription
So far we have only talked about gene regulate before transcription, What about after…

53. Different mRNA’s can be made from the same gene by alternative splicing
Most mRNA in eukaryotes have several introns
If an exon were to be spliced out when two intons are cut this would create an new protein
This is called alternative splicing and is a deliberate way to create alternative proteins from a single gene

54. Alternative RNA splicing: allows different RNA molecules (and thus, different proteins) to be produced from same primary transcript (pre-mRNA)

55. Examples on page 221…
Before the human genome was sequenced…scientists believed there were 80,000 to 150,000 protein coding genes
They now know there are 24,000 genes
80 percent of this variation are due to alternative splicing
Example with humans and chimpanzees

56. Let’s review the kinds of RNA we’ve encountered so far:
mRNA
tRNA
rRNA
snRNP
And let’s add:
miRNA (micro RNA)(also known as RNAi) (Fire & Mello, Nobel Prize 2006)
siRNA (small interfering RNA)

57. MicroRNAs are important regulators of gene expression
Only a fraction of genes code for proteins
Biologists used to think that the rest of the genome was not transcribed and they called it “junk” DNA
They now know that some of it is transcribed into very small RNA molecules called microRNA (miRNA)
These usually code for genes that turn on or off gene expression (example page 222)

58. mRNA degradation
Nova clip RNAi

59. The microRNA targets a mRNA and inhibits its translation
It than degrades the target mRNA
This is a gene silencing mechanism

60. Translation of mRNA can be regulated
For most genes mRNA doesn’t necessarily relate to the number of proteins
A lot of mRNA doesn’t necessarily mean a lot of protein or vice versa
Cells regulate how many proteins get made after transcription in 2 ways:
Regulation the translation of mRNA
Altering how long proteins persist in the cell

61. 3 ways in which translation can be regulated (examples page 223)
1. Inhibition of translation with miRNAs
2. Modification of the 5’ cap
An mRNA that is capped with an unmodified GTP molecule is not translated
3. Translational repressor proteins
Block translation by binding to mRNAs and preventing their attachment to the ribosome
Known as translational repressors

62. Protein stability can be regulated
Protein content is a result of both protein synthesis and protein degradation
Proteins can be targeted for destruction in a chain of events that begins when an enzyme attaches a protein called ubiquitin
Other ubiquitins then attach to the primary one forming a polyubiquitin

64. The polyubiquitin then binds to a protein complex called a proteasome
This then unfolds the protein inside of it
Example the breakdown of cyclins during the cell cycle
This needs to be done at the correct time and it is done by proteasomes

66. Cyclin-dependent kinase,
phosphorylates substrate proteins
Maturation-promoting factor (or M-phase PF)