1. CHAPTER 16
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2. Control of Gene Expression
Controlling gene expression is often accomplished by controlling transcription initiation
Regulatory proteins bind to DNA
May block or stimulate transcription
Prokaryotic organisms regulate gene expression in response to their environment
Eukaryotic cells regulate gene expression to maintain homeostasis in the organism

3. Regulatory Proteins
Gene expression is often controlled by regulatory proteins binding to specific DNA sequences
Regulatory proteins gain access to the bases of DNA at the major groove
Regulatory proteins possess DNA-binding motifs

4. DNA-binding motifs Regions of regulatory proteins which bind to DNA
Helix-turn-helix motif
Homeodomain motif
Zinc finger motif
Leucine zipper motif

5. 5 a. b. The Helix-Turn-Helix Motif The Leucine Zipper Motif α Helix
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The Helix-Turn-Helix Motif
The Leucine Zipper Motif
α Helix
(Recognition helix)
Turn
Zipper region
α Helix
Turn
α Helix
3.4 nm
90° a. b. 5

6. Prokaryotic regulation
Control of transcription initiation
Positive control – increases frequency of initiation of transcription
Activators enhance binding of RNA polymerase to promoter
Effector molecules can enhance or decrease
Negative control – decreases frequency
Repressors bind to operators in DNA
Allosterically regulated
Respond to effector molecules – enhance or abolish binding to DNA

7. Prokaryotic cells often respond to their environment by changes in gene expression
Genes involved in the same metabolic pathway are organized in operons
Induction – enzymes for a certain pathway are produced in response to a substrate
Repression – capable of making an enzyme but does not

8. Glucose repression
Preferential use of glucose in the presence of other sugars
Mechanism involves activator protein that stimulates transcription
Catabolic activator protein (CAP) is an allosteric protein with cAMP as effector
Level of cAMP in cells is reduced in the presence of glucose so that no stimulation of transcription from CAP-responsive operons takes place
Inducer exclusion – presence of glucose inhibits the transport of lactose into the cell

9. Eukaryotic Regulation
Control of transcription more complex
Major differences from prokaryotes
Eukaryotes have DNA organized into chromatin
Complicates protein-DNA interaction
Eukaryotic transcription occurs in nucleus
Amount of DNA involved in regulating eukaryotic genes much larger

10. Transcription factors
General transcription factors
Necessary for the assembly of a transcription apparatus and recruitment of RNA polymerase II to a promoter
TFIID recognizes TATA box sequences
Specific transcription factors
Increase the level of transcription in certain cell types or in response to signals

11. Promoters form the binding sites for general transcription factors
Mediate the binding of RNA polymerase II to the promoter
Enhancers are the binding site of the specific transcription factors
DNA bends to form loop to position enhancer closer to promoter

12. Coactivators and mediators are also required for the function of transcription factors
Bind to transcription factors and bind to other parts of the transcription apparatus
Mediators essential to some but not all transcription factors
Number of coactivators is small because used with multiple transcription factors

13. Transcription complex
Few general principles
Nearly every eukaryotic gene represents a unique case
Great flexibility to respond to many signals
Virtually all genes that are transcribed by RNA polymerase II need the same suite of general factors to assemble an initiation complex

15. Eukaryotic chromatin structure
Structure is directly related to the control of gene expression
DNA wound around histone proteins to form nucleosomes
Nucleosomes may block access to promoter
Histones can be modified to result in greater condensation

16. Methylation once thought to play a major role in gene regulation
Many inactive mammalian genes are methylated
Lesser role in blocking accidental transcription of genes turned off
Histones can be modified
Correlated with active versus inactive regions of chromatin
Can be methylated – found in inactive regions
Can be acetylated – found in active regions

17. Some coactivators have been shown to be histone acetylases
Transcription is increased by removing higher order chromatin structure that would prevent transcription
“Histone code” postulated to underlie the control of chromatin structure

19. Chromatin-remodeling complexes
Large complex of proteins
Modify histones and DNA
Also change chromatin structure
ATP-dependent chromatin remodeling factors
Function as molecular motors
Catalyze 4 different changes in DNA/histone binding
Make DNA more accessible to regulatory proteins

20. Pi ATP ADP + ATP -dependent remodeling factor 1. Nucleosome sliding
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ATP
ADP + Pi
ATP -dependent
remodeling factor
1. Nucleosome sliding
2. Remodeled nucleosome
3. Nucleosome displacement
4. Histone replacement

21. Posttranscriptional Regulation
Control of gene expression usually involves the control of transcription initiation
Gene expression can be controlled after transcription with
Small RNAs
miRNA and siRNA
Alternative splicing
RNA editing
mRNA degradation

22. Micro RNA or miRNA
Production of a functional miRNA begins in the nucleus
Ends in the cytoplasm with a ~22 nt RNA that functions to repress gene expression
miRNA loaded into RNA induced silencing complex (RISC)
RISC is targeted to repress the expression of genes based on sequence complementarity to the miRNA

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RNA Polymerase II
RNA Polymerase II
microRNA gene
microRNA gene
Pri-microRNA
Pri-microRNA
Nucleus
Nucleus
Drosha
Pre-microRNA
Pre-microRNA
Drosha
Exportin 5
Exportin 5
Cytoplasm
Dicer
Mature miRNA
RISC
mRNA
RISC
mRNA cleavage
mRNA
RISC
RISC 23
Inhibition of translation

24. siRNA RNA interference involves the production of siRNAs
Production similar to miRNAs but siRNAs arise from long double-stranded RNA
Dicer cuts yield multiple siRNAs to load into RISC
Target mRNA is cleaved

25. miRNA or siRNA?
Biogenesis of both miRNA and siRNA involves cleavage by Dicer and incorporation into a RISC complex
Main difference is target
miRNA repress genes different from their origin
Endogenous siRNAs tend to repress genes they were derived from

26. Repeated cutting by dicer
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Exogenous dsRNA, transposon, virus
Repeated cutting
by dicer P P P P P
siRNAs P P P
siRNA
in RISC
Ago +
Ago
RISC
RISC
mRNA
Cleavage of target mRNA

27. Alternative splicing
Introns are spliced out of pre-mRNAs to produce the mature mRNA
Tissue-specific alternative splicing
Same gene makes calcitonin in the thyroid and calcitonin-gene related peptide (CGRP) in the hypothalamus
Determined by tissue-specific factors that regulate the processing of the primary transcript

29. RNA editing
Creates mature mRNA that are not truly encoded by the genome
Involves chemical modification of a base to change its base-pairing properties
Apolipoprotein B exists in 2 isoforms
One isoform is produced by editing the mRNA to create a stop codon
This RNA editing is tissue-specific

30. Initiation of translation can be controlled
Ferritin mRNA only translated if iron present
Mature mRNA molecules have various half-lives depending on the gene and the location (tissue) of expression
Target near poly-A tail can cause loss of the tail and destabilization

31. Primary RNA transcript Mature RNA transcipt
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RNA polymerase II
1. Initiation of
transcription
Most control of
gene expression
is achieved
by regulating
the frequency
of transcription
initiation.
Cut
intron
2. RNA splicing
Gene expression
can be controlled
by altering the
rate of splicing in
eukaryotes.
Alternative splicing
can produce
multiple mRNAs
from one gene.
DNA 3´
3´ poly-A tail
5´ cap 5´
Primary RNA transcript
Exons
Introns
Mature RNA transcipt

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Large
subunit
3. Passage through
the nuclear
membrane
Gene expression
can be regulated
by controlling
access to or
efficiency of
transport channels.
3´ poly-A tail
Nuclear
pore
mRNA
5´ cap
Small
subunit
4. Protein synthesis
Many proteins take
part in the
translation process,
and regulation
of the availability
of any of them alters
the rate of gene
expression by
speeding or slowing
protein synthesis. 3´ 5´

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Completed
polypeptide
chain
6. Posttranslational
modification
Phosphorylation
or other chemical
modifications can
alter the activity
of a protein after
it is produced.
5. RNA interference
Gene expression
is regulated by
small RNAs. Protein
complexes
containing siRNA
and miRNA target
specific mRNAs for
destruction or inhibit
their translation. P
RISC P

34. Protein Degradation
Proteins are produced and degraded continually in the cell
Lysosomes house proteases for nonspecific protein digestion
Proteins marked specifically for destruction with ubiquitin
Degradation of proteins marked with ubiquitin occurs at the proteasome