Gene expression and regulation

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

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.

cis-acting

trans-acting

Regulatory elements

Promoter Enhancer Silencer Attenuator Terminator Insulators (boundary elements ) Response elements Transcription factor

Promoter

a combination of short sequence elements to which RNA polymerase

binds in order to initiate transcription of a gene

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

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

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

Structures of eukaryotic promoters

The promoter has three components: consensusSequences at -35 and -10, and the startpoint in E. Coli

In prokaryotic cells

Promoters recognized by E. coli RNA polymerase containing s70

Eukaryotic promoters consist of a collection of conserved short sequence elements located at relatively constant distances from

the transcription start site

Comparison of nucleotide sequences upstream of the start site in 60 different vertebrate protein-coding genes

Conserved locations in complex eukaryotes for regulatory promoter elements bound by ubiquitous transcription factors

INR: initiator; BRE: TFIIB recognition element; DPE: downstream promoter element;

General pattern of cis-acting control elements that regulate gene expression in yeast and multicellular organisms (invertebrates,

vertebrates, and plants)

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

Identification of SV40 enhancer region

←

←

The SV40 enhancer

The immunoglobulin enhancer

Action of enhancers

DNA looping

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

Life cycle of S. cerevisiae

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

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

trp operon

Attenuation provides a secondary mechanism for controlling expression of the trp operon

Terminator

A sequences of DNA that causes RNA polymerase to terminate transcription, e.g. AATAAA with an inverted palindrome

Rho-dependent termination

Rho is a helicase

Sequence of trp termination (t) site, a Rho-independent site

Termination at an intrinsic terminator

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 )

Schematic diagram summarizing the properties of insulators

Mechanism of imprinting of the mouse Igf2 gene

Localization of a Drosophila insulator-binding protein on polytene chromosomes

Response elements

sequence usually located a short distance upstream of promoters that makes gene expression responsive to some chemical in the cellular environment

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

Steroid receptors and the respective response elements

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

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

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

The molecular apparatus controlling transcription in human cells consists of four kinds of components

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

Structure of transcriptional activators

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

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

homeodomain A type of DNA binding domain found in transcription

factors that regulate gene expression during embryonic development

Structural motifs commonly found in transcription factors and DNA-binding proteins

Families of DNA-binding domains

Synergistic action of transcriptional activators

Eukaryotic Repressors eukaryotic repressors bind to specific DNA

sequences and inhibit transcription.

Action of eukaryotic repressors

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

Six steps at which eucaryotic gene expression can be controlled

Overview of transcription control in multicellular eukaryotes

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

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

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

DNA gyrase

Molecular model for the catalytic activity of E. coli topoisomerase II (DNA gyrase)

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

Histone acetylation

DNA methylation

A model for the structure of an interphase chromosome

Structure of a chromatosome

Chromatin fibers

Histone acetylation

DNA methylation

DNA methylation is capable of inhibiting transcription

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

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

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-

Regulation of mRNA Processing

---Splicing and Polyadenylation

Tissue-Specific RNA Splicing Controls

RNA Splicing of alternative exons

Alternative patterns of RNA splicing

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

Two examples of complex eukaryotic transcription units

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

Expression of Sex-lethal (Sxl) protein during Drosophila embryogenesis

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

Regulation of the site of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound

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

RNA editing of apo-B pre-mRNA

RNA editing Unusual type of RNA processing in which the sequence of a pre-mRNA is altered

Mechanism of RNA editing in kinetoplast pre-mRNAs of trypanosomes

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

Pathways for degradation of eukaryotic mRNA

Experimental demonstration of the destabilizing effect of AUUUA sequences on mRNA half-life (t1/2)

Iron-dependent regulation of the stability of transferrin-receptor (TfR) mRNA

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.

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

Insulin synthesis involves multiple post-translational cleavages of polypeptide precursors

Intrachain and interchain disulfide bridges in human insulin

References

Gene VIII Human molecular genetics 2 Molecular cell biology Molecular biology of the cell Genomes Modern Genetic Analysis

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