Control of Gene Expression Chapter 16. 2 DNA RNA Protein replication (mutation!) transcription...

Control of Gene ExpressionChapter 16

2

DNADNA

RNARNA

ProteinProtein

replicationreplication (mutation!)(mutation!)

transcriptiontranscription

translationtranslation

(nucleotides)

(amino acids)

(nucleotides)

Nucleic acids ~“software”

genes

messages

“hardware”

nucleus

ribosome(cytoplasm)

5

Fig. 16.1

6

Helix-Turn-Helix Motif

7

Homeodomain Motif

8

Zinc Finger Motif

9

Leucine Zipper Motif

11

repressor

lactose

primary RNA transcript

LacZ LacY LacAoperatorpromoter

RNA-P

repressor

DNA

mRNA

start codon stop codon stop codon stop codon

protein products of the operon:

The Lac Operon:

beta-galactosidase

permease acetylase

LacI

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genotype B-gal Permeasephenotype for:

lacI+ Y+Z+

lacI+ Z+ Y+lacO+

+IPTG --IPTG +IPTG --IPTG

lacIc Y+Z+

lacIS Y+Z+

lacOc Y+Z+

lacI+ Y--Z--

lacIc Y+Z+

lacOc Y+Z+

lacO+ Y--Z--

lacI-- Y+Z+

lacI+ Y+Z+

lacI+ Y+Z+

lacIc Y--Z--

lacOc Y--Z+

lacO+ Y+Z--

lacI-- Z+ Y+lacOc

++ ----

++ ++---- ----++ ++++ ----

++ ----

---- ----

++ ++

++ --+

++ ++

Lac+

Lacc

Lac-

Lacc

Lac+

Lac+

Lac-

Lacc

Lacc/Lac+

Lacc

}}

}}

lacI+ dominantin cis and trans

lacI-- dominant

lacOc dominantin cis

lacOc dominantin cis even in

presenceof lacI--

Data of Jacob and Monod, 1961

13


lacI+ Y+Z+

lacI+ Z+ Y+lacO+


lacIc Y+Z+

lacIS Y+Z+

lacOc Y+Z+

lacI+ Y--Z--

lacIc Y+Z+

lacOc Y+Z+

lacO+ Y--Z--

lacI-- Y+Z+

lacI+ Y+Z+

lacI+ Y+Z+

lacIc Y--Z--

lacOc Y--Z+

lacO+ Y+Z--

lacI-- Z+ Y+lacOc

++ ----

++ ++---- ----++ ++++ ----

++ ----

---- ----

++ ++

++ --+

++ ++

Lac+

Lacc

Lac-

Lacc

Lac+

Lac+

Lac-

Lacc

Lacc/Lac+

Lacc

}}

}}


lacI-- dominant



presenceof lacI--


14


lacI+ Y+Z+

lacI+ Z+ Y+lacO+


lacIc Y+Z+

lacIS Y+Z+

lacOc Y+Z+

lacI+ Y--Z--

lacIc Y+Z+

lacOc Y+Z+

lacO+ Y--Z--

lacIS Y+Z+

lacI+ Y+Z+

lacI+ Y+Z+

lacIc Y--Z--

lacOc Y--Z+

lacO+ Y+Z--

lacI-- Z+ Y+lacOc

++ ----

++ ++---- ----++ ++++ ----

++ ----

---- ----

++ ++

++ --+

++ ++

Lac+

Lacc

Lac-

Lacc

Lac+

Lac+

Lac-

Lacc

Lacc/Lac+

Lacc

}}

}}


lacIS dominant



presenceof lacI--


15


lacI+ Y+Z+

lacI+ Z+ Y+lacO+


lacIc Y+Z+

lacIS Y+Z+

lacOc Y+Z+

lacI+ Y--Z--

lacIc Y+Z+

lacOc Y+Z+

lacO+ Y--Z--

lacIS Y+Z+

lacI+ Y+Z+

lacI+ Y+Z+

lacIc Y--Z--

lacOc Y--Z+

lacO+ Y+Z--

lacI-- Z+ Y+lacOc

++ ----

++ ++---- ----++ ++++ ----

++ ----

---- ----

++ ++

++ --+

++ ++

Lac+

Lacc

Lac-

Lacc

Lac+

Lac+

Lac-

Lacc

Lacc/Lac+

Lacc

}}

}}


lacIS dominant



presenceof lacI--


16


lacI+ Y+Z+

lacI+ Z+ Y+lacO+


lacIc Y+Z+

lacIS Y+Z+

lacOc Y+Z+

lacI+ Y--Z--

lacIc Y+Z+

lacOc Y+Z+

lacO+ Y--Z--

lacIS Y+Z+

lacI+ Y+Z+

lacI+ Y+Z+

lacIc Y--Z--

lacOc Y--Z+

lacO+ Y+Z--

lacIS Z+ Y+lacOc

++ ----

++ ++---- ----++ ++++ ----

++ ----

---- ----

++ ++

++ --+

++ ++

Lac+

Lacc

Lac-

Lacc

Lac+

Lac+

Lac-

Lacc

Lacc/Lac+

Lacc

}}

}}


lacIS dominant



presenceof lacIS


23

Fig. 16.7

24From: http://www.aw.com/mathews/ch26/c26lara.htm

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Attenuation of trp operon transcription

28

Fig. 16.8

35

Fig. 16.16

40

Control of Gene Expression

• Controlling gene expression is often accomplished by controlling transcription initiation.

• Regulatory proteins bind to DNA to either block or stimulate transcription, depending on how they interact with RNA polymerase.

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Control of Gene Expression

• Prokaryotic organisms regulate gene expression in response to their environment.

• Eukaryotic cells regulate gene expression to maintain homeostasis in the organism.

42

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

43

Regulatory Proteins

• DNA-binding motifs are regions of regulatory proteins which bind to DNA

– helix-turn-helix motif

– homeodomain motif

– zinc finger motif

– leucine zipper motif

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Prokaryotic Regulation

• Control of transcription initiation can be:

– positive control – increases transcription when activators bind DNA

– negative control – reduces transcription when repressors bind to DNA regulatory regions called operators

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• Prokaryotic cells often respond to their environment by changes in gene expression.

• Genes involved in the same metabolic pathway are organized in operons.

• Some operons are induced when the metabolic pathway is needed.

• Some operons are repressed when the metabolic pathway is no longer needed.

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• The lac operon contains genes for the use of lactose as an energy source.

• Regulatory regions of the operon include the CAP binding site, promoter, and the operator.

• The coding region contains genes for 3 enzymes: -galactosidase, permease, and

transacetylase

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• 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 binds to the repressor protein

– repressor can no longer bind to operator

– transcription proceeds

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• In the presence of both glucose and lactose, bacterial cells prefer to use glucose.

• Glucose prevents induction of the lac operon.

– binding of CAP – cAMP complex to the CAP binding site is required for induction of the lac operon

– high glucose levels cause low cAMP levels

– high glucose low cAMP no induction

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• The trp operon encodes 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.

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• 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

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Eukaryotic Regulation

• Controlling the expression of eukaryotic genes requires transcription factors.– general transcription factors are

required for transcription initiation• required for proper binding of RNA

polymerase to the DNA– specific transcription factors increase

transcription in certain cells or in response to signals

52

Eukaryotic Transcription

• General transcription factors bind to the promoter region of the gene.

• RNA polymerase II then binds to the promoter to begin transcription at the start site (+1).

• Enhancers are DNA sequences to which specific transcription factors (activators) bind to increase the rate of transcription.

53

Eukaryotic Transcription

• Coactivators and mediators are also required for the function of transcription factors.

– coactivators and mediators bind to transcription factors and bind to other parts of the transcription apparatus

54

Eukaryotic Chromosome Structure

• Eukaryotic DNA is packaged into chromatin.

• Chromatin structure is directly related to the control of gene expression.

• Chromatin structure begins with the organization of the DNA into nucleosomes.

• Nucleosomes may block RNA polymerase II from gaining access to promoters.

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Eukaryotic Chromosome Structure

• Methylation (the addition of –CH3) of DNA or histone proteins is associated with the control of gene expression.

• Clusters of methylated cytosine nucleotides bind to a protein that prevents activators from binding to DNA.

• Methylated histone proteins are associated with inactive regions of chromatin.

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Posttranscriptional Regulation

• Control of gene expression usually involves the control of transcription initiation.

• But gene expression can be controlled after transcription, with mechanisms such as:

– RNA interference

– alternative splicing

– RNA editing

– mRNA degradation

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• RNA interference involves the use of small RNA molecules

• The enzyme Dicer chops double stranded RNA into small pieces of RNA

– micro-RNAs bind to complementary RNA to prevent translation

– small interfering RNAs degrade particular mRNAs before translation

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• Introns are spliced out of pre-mRNAs to produce the mature mRNA that is translated.

• Alternative splicing recognizes different splice sites in different tissue types.

• The mature mRNAs in each tissue possess different exons, resulting in different polypeptide products from the same gene.

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• RNA editing creates mature mRNA that are not truly encoded by the genome.

• For example –

– 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

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• Mature mRNA molecules have various half-lives depending on the gene and the location (tissue) of expression.

• The amount of polypeptide produced from a particular gene can be influenced by the half-life of the mRNA molecules.

61

Protein Degradation

• Proteins are produced and degraded continually in the cell.

• Proteins to be degraded are tagged with ubiquitin.

• Degradation of proteins marked with ubiquitin occurs at the proteasome.

Control of Gene Expression Chapter 16. 2 DNA RNA Protein replication (mutation!) transcription...

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