Control of Gene Expression

1
Control of Gene Expression

• Chapter 16

Biology Dual Enrollment Mrs. Mansfield
1
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
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

6

• 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

7
lac operon

• Contains genes for the use of lactose as an
  energy source
• -b-galactosidase (lacZ), permease (lacY), and
  transacetylase (lacA)
• Gene for the lac repressor (lacI) is linked to
  the rest of the lac operon

8

• The lac operon is negatively regulated by a
  repressor protein
• lac repressor binds to the operator to block
  transcription
• In the presence of lactose, an inducer molecule
  (allolactose) binds to the repressor protein
• Repressor can no longer bind to operator
• Transcription proceeds
• Even in the absence of lactose, the lac operon is
  expressed at a very low level

9
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

10
trp operon

• Genes for the biosynthesis of tryptophan
• The operon is not expressed when the cell
  contains sufficient amounts of tryptophan
• The operon is expressed when levels of tryptophan
  are low
• trp repressor is a helix-turn-helix protein that
  binds to the operator site located adjacent to
  the trp promoter

11

• The trp operon is negatively regulated by the trp
  repressor protein
• trp repressor binds to the operator to block
  transcription
• Binding of repressor to the operator requires a
  corepressor which is tryptophan
• Low levels of tryptophan prevent the repressor
  from binding to the operator

12
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

13
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

14

• 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

15

• 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

16
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

17
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

18

• 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

19

• 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

20

• 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

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

23
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

24
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

25
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

26
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

27

• 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

28
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