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Gene expression and regulation
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cis-acting Referring to a regulatory sequence in DNA (e.g., enhancer, promoter) that can control a gene only on the same chromosome. In bacteria, cis-acting elements are adjacent or proximal to the gene(s) they control, whereas in eukaryotes they may also be far away
trans-acting Referring to DNA sequences encoding diffusible proteins or RNA (e.g., transcription activators, repressors and tRNA) that control genes on the same or different chromosomes
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transcription factor (TF) General term for any protein, other than RNA polymerase, required to initiate or regulate transcription in eukaryotic cells. General factors, required for transcription of all genes, participate in formation of the transcription-initiation complex near the start site. Specific factors stimulate (or repress) transcription of particular genes by binding to their regulatory sequences
transcription unit A region in DNA, bounded by an initiation (start) site and termination site, that is transcribed into a single primary transcript
transcription-control region Collective term for all the cis-acting DNA regulatory sequences that regulate transcription of a particular gene.
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cis-acting
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trans-acting
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Regulatory elements
Promoter Enhancer Silencer Attenuator Terminator Insulators (boundary elements ) Response elements Transcription factor
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Promoter
a combination of short sequence elements to which RNA polymerase
binds in order to initiate transcription of a gene
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In eukaryotic cells:
TATA box A conserved sequence in the promoter of many
eukaryotic protein-coding genes where the transcription-initiation complex assembles, located at -20~-30 with the sequence of TATAA(T)AA(T) which firstly binds with transcriptional factor TFII
CAAT box A basal promoter element located -70~-80 with
GGC(T)CAATCA where the transcriptional factor CTF binds with for increasing the efficiency of transcription
GC box A type of basal promoter element with GGCGGG
which can bind with transcriptional factor Sp1
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In prokaryotic cells:
-10 sequence The consensus sequence centered about 10 bp
before the startpoint of a bacterial gene. It is involved in melting DNA during the initiation reaction
-35 sequence The consensus sequence centered about 35 bp
before the startpoint of a bacterial gene. It is involved in initial recognition by RNA polymerase
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Promoters and terminators define a transcription unit in DNA, starting at the promoter and ending at the terminator
Startpoint (startsite): refers to the position on DNA corresponding to the first baseIncorporated into RNA
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Structures of eukaryotic promoters
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The promoter has three components: consensusSequences at -35 and -10, and the startpoint in E. Coli
In prokaryotic cells
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Promoters recognized by E. coli RNA polymerase containing s70
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Eukaryotic promoters consist of a collection of conserved short sequence elements located at relatively constant distances from
the transcription start site
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Comparison of nucleotide sequences upstream of the start site in 60 different vertebrate protein-coding genes
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Conserved locations in complex eukaryotes for regulatory promoter elements bound by ubiquitous transcription factors
INR: initiator; BRE: TFIIB recognition element; DPE: downstream promoter element;
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General pattern of cis-acting control elements that regulate gene expression in yeast and multicellular organisms (invertebrates,
vertebrates, and plants)
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Enhancer
a set of short sequence elements which stimulate transcription of a gene and whose function is not critically dependent on their precise position or orientation
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Identification of SV40 enhancer region
←
←
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The SV40 enhancer
The immunoglobulin enhancer
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Action of enhancers
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DNA looping
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Silencer
A sequence in eukaryotic DNA that promotes formation of condensed chromatin structures in a localized region, thereby blocking access of proteins required for transcription of genes within several hundred base pairs of the silencer sequence
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Life cycle of S. cerevisiae
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Silencer Elements Repress Expression at HML and HMR
Gene conversion Phenomenon in which one allele of a gene is converted to another during meiotic recombinationRecombination Any process in which chromosomes or DNA molecules are cleaved and the fragments are rejoined to give new combinations. Occurs naturally in cells as the result of the exchange (crossing over) of DNA sequences on maternal and paternal chromatids during meiosis; also is carried out in vitro with purified DNA and enzymes
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Attenuator
A region adjacent to the structural genes of the trp operon; this region acts in the presence of tryptophan to reduce the rate of transcription from the structural genes
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trp operon
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Attenuation provides a secondary mechanism for controlling expression of the trp operon
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Terminator
A sequences of DNA that causes RNA polymerase to terminate transcription, e.g. AATAAA with an inverted palindrome
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Rho-dependent termination
Rho is a helicase
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Sequence of trp termination (t) site, a Rho-independent site
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Termination at an intrinsic terminator
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Insulators
are regions of DNA, often spanning 0.5~3 kb, which function to block (or insulate) the spreading of the influence of agents that have a positive effect on transcription (enhancers) or a negative one (silencers, heterochromatin-like repressive effects )
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Schematic diagram summarizing the properties of insulators
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Mechanism of imprinting of the mouse Igf2 gene
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Localization of a Drosophila insulator-binding protein on polytene chromosomes
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Response elements
sequence usually located a short distance upstream of promoters that makes gene expression responsive to some chemical in the cellular environment
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Consensus response element (RE)
Response to: Protein factor which recognizes RE
(T/G)(T/A)CGTCA cAMP CREB (also called ATF)
CC(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)GG
Serum growth factor
Serum response factor
TTNCNNNAAA Interferon-gamma
Stat-1
TGCGCCCGCC Heavy metals Mep-1
TGAGTCAG Phorbol esters
AP1
CTNGAATNTTCTAGA Heat shock HSP70, etc.
Examples of response elements in inducible gene expression
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Steroid receptors and the respective response elements
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Transcription factor
A protein that regulates the activity of RNA polymerase
Transcription of eukaryotic genes requires interaction between cis-acting transcription elements and trans-acting transcription factors
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Cis element DNA sequence is identical to, or a variant of
Associated trans-acting factors
Comments
GC box GGGCGG Spl Spl factor is ubiquitous
TATA box TATAAA TFIID TFIIA binds to the TFIID-TATA box complex to stabilize it
CAAT box CCAAT Many, e.g. C/EBP, CTF/NFI
Large family of trans-acting factors
CRE (cAMP response element)
GTGACGTA/CAA/G
CREB/ATF family, e.g. ATF-1
Genes activated in response to cAMP
Examples of cis-acting elements recognized by ubiquitous transcription factors
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Consensus binding sequence
Transcription factor
Expression patterns
(A/T)GATA(A/G) GATA-1, -2, etc.
Erythroid cells
TGACTCAG NF-E2 Erythroid cells
GTTAATNATTAAC (= PE element)
HNF-1 Differentiated liver, kidney, stomach, intestine, spleen
T(G/A)TTTG(C/T) HNF-5 Liver
GCCTGCAGGC Ker1 Keratinocytes
(C/T)TAAAAATAA(C/T)3 MBF-1 Myocytes
(C/T)TA(A/T)AAATA(A/G) MEF-2 Myocytes
CAACTGAC MyoD Myoblasts + myotubes
ATGCAAAT OTF-2 Lymphoid cells
(C/A)A(C/A)AG TCF-1 T cells
Examples of cis-acting sequences recognized by tissue-restricted and tissue-specific transcription factors
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The molecular apparatus controlling transcription in human cells consists of four kinds of components
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An activation domain to be rich in aspartate and glutamate
residues (acidic activation domains); others are rich
in proline or glutamate A DNA-binding domain ---The leucine zipper motif
---The helix-loop-helix motif
---The helix-turn-helix motif ---The zinc finger motif ---Homeodomain
Structure and Function of Transcriptional ActivatorsStructure and Function of Transcriptional Activators
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Structure of transcriptional activators
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Some concepts
zinc finger Several types of conserved DNA-binding motifs composed of protein domains containing cysteine and histidine residues that bind zinc ions; present in several types of eukaryotic transcription factors
leucine zipper Common structural motif in some dimeric eukaryotic transcription factors
characterized by a C-terminal coiled-coil dimerization domain and N-terminal DNA-binding domain containing repeated leucine residues; found in many transcription factors
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helix-loop-helix A conserved structural motif found in many monomeric Ca2+-binding proteins and dimeric eukaryotic transcription factors
helix-turn-helix It consists of two short α-helices separated by a short amino acid sequence which induces a turn, so that the two α-helices are orientated differently
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homeodomain A type of DNA binding domain found in transcription
factors that regulate gene expression during embryonic development
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Structural motifs commonly found in transcription factors and DNA-binding proteins
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Families of DNA-binding domains
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Synergistic action of transcriptional activators
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Eukaryotic Repressors eukaryotic repressors bind to specific DNA
sequences and inhibit transcription.
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Action of eukaryotic repressors
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The regulation of gene expression
1. Transcriptional control2. Post-transcriptional control RNA processing control RNA transport and localization
control translational control mRNA degradation control protein activity control 3. Epigenetic mechanisms and long
range control of gene expression
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Six steps at which eucaryotic gene expression can be controlled
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Overview of transcription control in multicellular eukaryotes
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Selective expression mechanism Transcriptional
Binding of tissue-specific transcription factors to cis-acting elements of a single gene
Direct binding of hormones, growth factors or intermediates to response elements in inducible transcription elements
Use of alternative promoters in a single gene
Post-transcriptional
Alternative splicing
Alternative polyadenylation
Tissue-specific RNA editing
Translational control mechanisms
Epigenetic mechanisms and long-range control of gene expression by chromatin structure
Allelic exclusion is expected to embrace four example
Long-range control by chromatin structure
Overview of the regulation of gene expression in human cells
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Transcriptional Control
Trans-acting factors (protein or maybe RNA) affect expression of all copies of a gene cis-acting elements (normally DNA sequences) regulate only the DNA molecule of which they are part
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Class Genes transcribed Comments
I 28S rRNA; 18S rRNA; 5.8S rRNA
Localized in the nucleolus. A single primary transcript (45S rRNA) is cleaved to give the three rRNA classes listed
II All genes that encode polypeptides; most snRNA genes
Polymerase II transcripts are unique in being subject to capping and polyadenylation
III 5S rRNA; tRNA genes; U6 snRNA; 7SL RNA; 7SK RNA; 7SM RNA
The promoter for some genes transcribed by RNA polymerase III (e.g. 5S rRNA, tRNA, 7SL RNA) is internal to the gene and for others (e.g. 7SK RNA) is located upstream
The three classes of eukaryotic RNA polymerase
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DNA gyrase
Molecular model for the catalytic activity of E. coli topoisomerase II (DNA gyrase)
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TypeType SubstrateSubstrate ExamplesExamples
Type Type IAIA
Single-Single-stranded DNAstranded DNA
Escherichia coliEscherichia coli topoisomerases I and III; yeast and human topoisomerases I and III; yeast and human topoisomerase III; archaeal reverse gyrasetopoisomerase III; archaeal reverse gyrase
Type Type IBIB
Single-Single-stranded DNAstranded DNA
Eukaryotic topoisomerase IEukaryotic topoisomerase I
Type Type IIII
Double-Double-stranded DNAstranded DNA
E. coliE. coli topoisomerases II (DNA gyrase) and IV; eukaryotic topoisomerases II (DNA gyrase) and IV; eukaryotic topoisomerases II and IVtopoisomerases II and IV
DNA topoisomerases
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Histone acetylation
DNA methylation
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A model for the structure of an interphase chromosome
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Structure of a chromatosome
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Chromatin fibers
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Histone acetylation
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DNA methylation
DNA methylation is capable of inhibiting transcription
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Transcriptional repression by histone deacetylation may be mediated by DNA methylation
methylated CpGs are targets for specific binding by proteins such as MeCP2 (methylated CpG binding proteins2), which acts as a transcriptional repressor and recruits a corepressor complex consisting of the transcription factor repressor mSin3A and histone deacetylases
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Feature Transcriptionally active chromatin
Transcriptionally inactive chromatin
Chromatin conforma-tion
Open, extended conformation
Highly condensed conformation; particularly apparent in heterochromatin (both facultative and constitutive; see Section 3.5)
DNA methylation
Relatively unmethylated, especially at promoter regions
Methylated, including at promoter regions
Histone acetylation
Acetylated histones Deacetylated histones
Features associated with transcriptionally active and inactive chromatin
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Copyright ©2006 by the National Academy of Sciences
Gomis, Roger R. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 12747-12752
Variability in promoter configuration and regulation in the FoxO-Smad synexpression group in response to TGF-
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Regulation of mRNA Processing
---Splicing and Polyadenylation
Tissue-Specific RNA Splicing Controls
RNA Splicing of alternative exons
Alternative patterns of RNA splicing
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The major human rRNA species are synthesized by cleavage from a common 13 kb transcription unit which is part of a 40 kb tandemly repeated unit
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Two examples of complex eukaryotic transcription units
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Cell type-specific splicing of fibronectin pre-mRNA in fibroblasts and hepatocytes
(Exons, EIIIA and EIIIB, encode protein domains that interact with cell-surface receptors in many cell types)
tissue-specific RNA splicing
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Expression of Sex-lethal (Sxl) protein during Drosophila embryogenesis
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Alternative splicing of slo mRNA, which encodes a Ca2+ -gated K+ Channel,
in auditory hair cells contributes to the preception of sounds of
different frequency
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Regulation of the site of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound
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Other Mechanisms of Post-Transcriptional Control
---RNA Editing Alters the Sequences of Pre-mRNAs
---Some mRNAs Are Associated with Cytoplasmic Structures or Localized to Specific Regions
---Stability of Cytoplasmic mRNAs Varies Widely ---Degradation Rate of Some Eukaryotic mRNAs Is
Regulated ---Translation of Some mRNAs Is Regulated by
Specific RNA-Binding Proteins ---Antisense RNA Regulates Translation of
Transposase mRNA in Bacteria
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RNA editing of apo-B pre-mRNA
RNA editing Unusual type of RNA processing in which the sequence of a pre-mRNA is altered
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Mechanism of RNA editing in kinetoplast pre-mRNAs of trypanosomes
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mRNA Half-Lives
Cell Cell Generation Time
Average Range Known for Individual Cases
Escherichia coli 20~60 min 3~5 min 2~10 min
Saccharomyces cerevisiae (yeast)
3 h 22 min 4~40 min
Cultured human or rodent cells
16~24 h 10 h 30 min or less
(histone and c-myc mRNAs);
0.3~24 h (specific mRNAs of
cultured cells)
Half-Lives of Messenger RNAs
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Pathways for degradation of eukaryotic mRNA
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Experimental demonstration of the destabilizing effect of AUUUA sequences on mRNA half-life (t1/2)
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Iron-dependent regulation of the stability of transferrin-receptor (TfR) mRNA
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Translational controlPost-translational modifications of proteins are frequent and can involve addition of specific chemical groups to specific amino acids and cleavage of the primary translation product
* Protein modification by addition of carbohydrate groups N-glycosylation O-glycosylation
* Protein modification by addition of lipid groups membrane proteins, are modified by the addition of fatty acyl or prenyl groups which typically serve as membrane anchors
* Post-translational cleavage Signal sequence (leader sequence) a sequence of about 20 aminoacids at the N-terminus of a polypeptide that controls its destination within or outside the cell.
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Type of modification
(group added)
Target amino acids Comments
Phosphorylation Tyrosine, serine, threonine
(PO4-) Achieved by specific kinases. May be reversed by phosphatases
Methylation (CH3) Lysine Achieved by methylases and undone by demethylases
Hydroxylation (OH) Proline, lysine, aspartic acid
Hydroxyproline and hydroxylysine are particularly common in collagens
Acetylation (CH3CO) Lysine Achieved by an acetylase and undone by deacetylase
Farnesylation (C15 prenyl group)
Cysteine at C terminus Serves as membrane anchor
Carboxylation (COOH) Glutamate Achieved by g-carboxylase
N-glycosylation (complex carbohydrate)
Asparagine, usually in the sequence: Asn-X-Ser/Thr
Takes place initially in the endoplasmic reticulum; X is any amino acid other than proline
O-glycosylation (complex carbohydrate)
Serine, threonine, hydroxylysine
Takes place in the Golgi apparatus; less common than N-glycosylation
Major types of modification of polypeptides
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Insulin synthesis involves multiple post-translational cleavages of polypeptide precursors
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Intrachain and interchain disulfide bridges in human insulin
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References
Gene VIII Human molecular genetics 2 Molecular cell biology Molecular biology of the cell Genomes Modern Genetic Analysis
E-mail: [email protected]