1. Chapter 9 Molecular Mechanisms of Gene Regulation

2. Transcription Regulation: Prokaryotes
In prokaryotes, on–off gene activity is often controlled through transcription
In negative regulation, the default state of transcription is “on” until it is turned “off” by a repressor protein that binds to the DNA upstream from the transcriptional start site

3. Transcription Regulation: Prokaryotes
In inducible transcription, a repressor keeps transcription in the “off” state (repressor + inducer  on)
In repressible transcription, the default state is “on” until an active repressor is formed to turn it “off” (aporepressor + co-repressor  off)

4. Figure 9.1: Negative regulation

5. Transcription Regulation: Prokaryotes
In positive regulation, the default state of transcription is “off” and binding with a transcriptional activator protein is necessary to turn it “on”
Many systems are both positively and negatively regulated
Negative regulation is more common in prokaryotes, positive regulation in eukaryotes
Some genes exhibit autoregulation—the protein product of a gene regulates its own transcription

6. Figure 9.2: Transcription

7. Operon Model of Regulation
In bacterial systems, when several enzymes act in sequence in a single metabolic pathway, usually either all or none of the enzymes are produced
This coordinate regulation results from control of the synthesis of one or more mRNA molecules that are polycistronic
Polycistronic mRNAs are transcribed from an operon: a collection of adjacent structural genes regulated by an operator

8. lac Operon
The genetic regulatory mechanism in bacteria was first explained by the operon model of François Jacob and Jacques Monod
They studied lactose-utilization system in E.coli
The lactose-utilization system consists of two kinds of components: structural genes (lacZ and lacY), which encode proteins needed for the transport and metabolism of lactose, and regulatory elements (the repressor gene lacI, the promoter lacP, and the operator lacO)

9. lac Operon
The products of the lacZ (enzyme b-galactosidase) and lacY (transporter lactose permease) genes are coded by a single polycistronic mRNA
The linked structural genes, together with lacP and lacO, constitute the lac operon
The promoter mutations (lacP–) eliminate the ability to synthesize lac mRNA

10. lac Operon
The product of the lacI gene is a repressor, which binds to a unique sequence of DNA bases constituting the operator
When the repressor is bound to the operator, initiation of transcription of lac mRNA by RNA polymerase is prevented
Because the repressor is necessary to shut off mRNA synthesis, regulation by the repressor is negative regulation

11. lac Operon
Lactose (inducer) stimulate mRNA synthesis by binding to and inactivating the repressor
Figure 9.4a/b: lac operon

12. lac Operon
In the presence of an inducer, the operator is not bound with the repressor, and the promoter is available for the initiation of mRNA synthesis
Figure 9.4c: lac operon

13. lac Operon lac operon is also subject to positive regulation
Positive regulation of the lac operon involves cAMP-CRP (cyclic AMP receptor protein), which binds to the promoter to activate transcription by RNA polymerase
In the absence of the cAMP–CRP complex, RNA polymerase binds only weakly to the promoter

14. Figure 9.6: Four regulatory states of the lac operon

15. trp Operon
The trp operon contains the structural genes that encode enzymes required for the synthesis of the amino acid tryptophan
The trp operon is transcriptionally active unless tryptophan is present
The trp operon is an example of a repressible system regulated by a negative feedback loop

16. Figure 9.9: The trp operon in E. coli

17. trp Operon
The trp operon is shut off when tryptophan binds to inactive aporepressor
Tryptophan-repressor complex binds to operator to block transcription when tryptophan levels are high
If tryptophan levels fall, trp-repressor complex dissociates from operator

18. Figure 9.10: Regulation of the E. coli trp operon

19. Figure 9.11: The terminal region of the trp attenuator sequence
Attenuation
Attenuation is a very sensitive form of translational
regulation of the trp operon
The trp attenuator consists of 5' base sequence in mRNA that is complementary and can base-pair to form a stem and loop structure
Figure 9.11: The terminal region of the trp attenuator sequence

20. Figure 9.12: The sequence of bases in the trp leader mRNA
Attenuation
Attenuation results in the premature termination of mRNA synthesis due to stem and loop formation in the 5' region of mRNA
If tRNATrp is present , synthesis of the leader peptide results in pairing of mRNA, which blocks the action of RNA polymerase
Figure 9.12: The sequence of bases in the trp leader mRNA

21. Figure 9.13: Mechanism of attenuation in the E. coli trp operon

22. Attenuation
At low concentrations of tRNATrp, the ribosome stalls, and the mRNA opens so that transcription continues
Attenuation permits the cell to respond to tryptophan levels by expressing the genes needed for its synthesis when needed
Attenuation is possible because in bacteria transcription is coupled with translation

23. Riboswitches
Transcription termination can also be triggered by direct binding of a small molecule (vitamin B12 or flavin mononucleotide) to a 5' untranslated leader mRNA
An RNA leader sequence able to switch between an antiterminator conformation and a terminator conformation is known as a riboswitch
Comparison of genomic sequences indicates that riboswitches are present in archaea, eubacteria, and eukarya

24. Gene Regulation: Eukaryotes
Gene regulation in eukaryotes differs from
prokaryotes:
The processes of transcription and translation are uncoupled
The primary transcripts of many genes are alternatively spliced to yield different products
Each gene is regulated by a separate, often multiple promoters

25. Gene Regulation: Eukaryotes
Genome organization in eukaryotes:
DNA in complex with histones forms chromatin
Significant fraction of DNA is moderately or highly repetitive
Large fraction of DNA does not code for proteins
Many coding sequences are interrupted by introns

26. Transcriptional Regulation
Many eukaryotic genes differ in their expression according to cell type or stage of the cell cycle. These genes are often regulated at the level of transcription.
Galactose metabolism in yeast illustrates transcriptional regulation

27. Figure 9.15: Metabolic pathway

28. Galactose Metabolism in Yeast
Despite the tight linkage of the three genes involved in galactose metabolism, they are not part of an operon. The mRNAs are monocistronic.
The GAL1 and GAL10 mRNAs are synthesized from divergent promoters lying between the genes, and GAL7 mRNA is synthesized from its own promoter.
These genes are inducible because the mRNAs are synthesized only when galactose is present.

29. Figure 9.16: The linked GAL genes of Saccharomyces cerevisiae

30. Galactose Metabolism in Yeast
Constitutive and uninducible mutants have been observed.
In two types of mutants, gal80 and gal81c, the mutants synthesize GAL1, GAL7, and GAL10 mRNAs constitutively.
Another type of mutant, gal4, is uninducible: It does not synthesize the mRNAs whether or not galactose is present.

31. Table 9.3: Characteristics of Diploids Containing Various Combinations of gal80, gal4, and gal81c Alleles

32. Enhancers
Enhancer sequences are typically rather short (~ 20 bp) and are found at a variety of locations around the gene they regulate
They may be located many kb from a gene or inside introns and may are able to function as enhancers irrespective of their orientation
Enhancers are essential components of gene organization in eukaryotes because they enable genes to be transcribed only when proper transcriptional activators are present

33. Silencers
Some genes are also subjected to regulation by transcriptional silencers: short nucleotide sequences that are targets for DNA-binding proteins that promote the assembly of large protein complexes that prevent transcription of the silenced genes
PcG (Polycomb group) proteins in D. melanogaster silence certain genes during development.

34. Chromatin-remodeling Complexes
Several different multiprotein complexes, known as chromatin-remodeling complexes (CRCs), can restructure chromatin and enable it to be transcribed
CRCs can disrupt nucleosome structure without displacing them or reposition the nucleosomes making key DNA-binding sites accessible
CRCs use energy derived from ATP to restructure chromatin
The molecular mechanism of chromatin remodeling is unknown

35. Figure 9.22a/b: Function of chromatin-remodeling complexes

36. Figure 9.22c/d: Function of chromatin-remodeling complexes

37. Alternative Promoters
Some eukaryotic genes have multiple promoters that are active in different cell types
The different promoters result in different primary transcripts that contain the same protein-coding regions
Alternative promoters make possible the independent regulation of transcription in different tissues or at different stages of development

38. Figure 9.23: Use of alternative promoters

39. Transcription Activation
Transcription complex is an aggregate of protein factors that combines with the promoter to initiate transcription
The basal transcription factors are proteins in the complex that are used in the transcription of many different genes
The basal transcription factors in eukaryotes have been highly conserved in evolution
Transcriptional activation occurs by a mechanism called recruitment—the interaction of transcription factors with promoter and enhancers

40. Figure 9.20: Transcriptional activation by recruitment

41. Transcription Regulation
Combinatorial control means that a few genes can control many others
Strategically placed enhancers can act as genetic switches
One gene can have two or more promoters that are regulated differently

42. Epigenetic Regulation
Epigenetic: heritable changes in gene expression that are due to either chemical modification of the DNA bases, or protein factors bound with the DNA
In most higher eukaryotes, a proportion of the cytosine base are modified by the addition of a methyl (CH3) group to the number-5 carbon atom
The cytosines are incorporated unmodified during DNA replication, and then the methyl group is added by an enzyme called DNA methylase

43. DNA Methylation
In mammals, cytosines are modified preferentially in 5'-CG-3' dinucleotides
Many mammalian genes have CG-rich regions upstream of the coding region that provide multiple sites for methylation—CpG islands
Heavy methylation is associated with genes for which the rate of transcription is low
One example is the inactive X chromosome in mammalian cells, which is extensively methylated.

44. Imprinting
Mammals feature an unusual type of epigenetic silencing known as genomic imprinting, a process with the following characteristics:
Imprinting occurs in the germ line and affects at most a few hundred genes
It is accompanied by heavy methylation
Imprinted genes are differentially methylated in the female and male germ lines
Once imprinted, a silenced gene remains inactive during embryogenesis
Imprints are erased early in germ-line development, then later reestablished according to sex-specific patterns

45. Imprinting
The epigenetic, sex-specific gene silencing associated with imprinting is dramatically evident in Prader–Willi syndrome and Angelman syndrome characterized by neuromuscular defects, mental retardation, and other abnormalities

46. Figure 9.24: Imprinting of genes and varying results

47. Alternative Splicing
Regulation also takes place at the levels of RNA processing
Alternative splicing is a form of gene regulation that results in the generation of alternative mRNAs from a single gene
Different splice patterns may occur in different tissues resulting in tissue-specific gene expression

48. Alternative Splicing
Alternative splicing is an important source of human genetic complexity
Although the number of human genes exceeds that in worms or flies by a factor of about two, the number of different human proteins may be greater than that in worms or flies by a factor of about five

49. Figure 9.25: Alternative splicing of the primary transcript

50. RNA Interference
Different mRNAs can differ in their persistence in the cell
An important mechanism regulating the stability of RNA transcripts is a phenomenon of RNA interference (RNAi), in which the introduction of a few hundred nucleotide pairs of double-stranded RNA triggers degradation of RNA transcripts containing homologous sequences

51. Figure 9.27: Mechanisms of gene silencing by the
siRNA and miRNA pathways

52. Translation Regulation
In eukaryotes, gene expression regulated at the level of translation separately from transcription
The principle types of translational control are:
Inability of an mRNA to be translated except under certain conditions
Regulation of the overall rate of protein synthesis
Inhibition or activation of translation by small regulatory RNAs that undergo base pairing with the mRNA

53. Figure 9.28: Regulation of translation of target mRNAs
Adapted from S. Altuvia and E. G. H. Wagner, Proc. Natl. Acad. Sci. USA 97 (2000):

54. Translation Regulation
An RNA sequence complementary to an mRNA is called an antisense RNA
The antisense regulatory RNAs act by pairing with the mRNA to either inhibit or activate translation
Regulatory RNAs that control developmental timing in the nematode C. elegans were the first examples of small regulatory RNAs described in eukaryotes.

55. DNA Rearrangements
Some developmental processes are controlled by programmed DNA rearrangements
A permanent change in DNA sequence implies that the genotype of a cell lineage becomes permanently altered
Such irreversible changes take place only in somatic cells, so they are not genetically transmitted
Programmed DNA rearrangement take place in the bone-marrow–derived (B) cells and thymus-derived (T) cells that play key roles in the vertebrate immune system

56. Figure 9.30: Formation of a gene for the light chain of an antibody molecule

57. DNA Rearrangements
Programmed transpositions take place in the regulation of yeast mating type
Many strains of S. cerevisiae have a mating system called homothallism in which some cells undergo a conversion into the opposite mating type
This allows matings between cells in what would otherwise be a pure culture

58. Figure 9.31: Genetic basis of mating-type interconversion