prokaryotic chromosome ( e. coli )
DESCRIPTION
Gene Expression Prokaryotic Gene Transcription (Cont.) & Eukaryotic Transcription Histones and Chromatin G & G Pages 336-340, Chapter 29, 9/14/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics [email protected]. Prokaryotic Chromosome ( E. coli ). Large circular chromosome - 4.6 x 10 6 bp - PowerPoint PPT PresentationTRANSCRIPT
Gene Expression
Prokaryotic Gene Transcription(Cont.)
&Eukaryotic Transcription
Histones and Chromatin
G & G Pages 336-340, Chapter 29,
9/14/11Thomas Ryan, Ph.D.
Biochemistry and Molecular [email protected]
Prokaryotic Chromosome (E. coli)
• Large circular chromosome - 4.6 x 106 bp
• Genome forms a compact structure called the nucleoid
• DNA organized in 50-100 loops (domains)
• The ends of loops are constrained by binding to protein structure which is in contact with cell membrane
Operators can be upstream, downstream, or overlapping with the promoter.
General Organization of Operons
Regulatory proteins that bind to the operator can influence the access of RNA polymerase to the promoter thereby affecting the rate of transcription initiation.
Induction and Repression• Increased synthesis of genes in response to
metabolites is ‘induction’ and the metabolites are called co-inducers
• Decreased synthesis in response to a metabolite is termed ‘repression’ and the metabolite a co-repressor
• Some substrates induce enzyme synthesis even though the enzymes can’t metabolize the substrate - these are ‘gratuitous inducers’ - such as IPTG
lac OperonBoth Positive and Negative Regulation
-35 -10
+1 mRNA
RNA Polymerase binding
Repressor binding-61.5
(CRP•cAMP)2
binding
Multiple control mechanisms are the norm.
lac Operon of E. coli
The lac OperonNegative regulation
• The structural genes of the lac operon are controlled by negative regulation
• lacI gene product is the lac repressor• lacI mutants express the genes needed for
lactose metabolism constitutively• The lac operator has palindromic DNA• lac repressor - forms tetramer; DNA binding in
the N-terminal domain; Inducer binding at the C-terminal domain
lac OperonlacI gene encodes a repressor
lac Operon Negative Regulation
•Operon is generally “off”; only fully “on” when lactose is present and glucose is absent
•When no lactose is present: repressor is bound; inhibiting transcription elongation through the operon
•When lactose is present: lactose is converted to allolactose, binds to repressor, causing it to fall off the operator and allowing transcription of the downstream cistrons to proceed
O
CH2OH
HO
OHO
O
OH
CH2OH
OH
OH
Lactose
-Galactoside linkage
O
CH2OHHO
OH
OH OH
S C H
CH3
CH3
(IPTG)
Isopropyl -D-thiogalactoside
substrate not a substrate
Gratuitous Inducers:
Not substrates, but still induce
glucose
O
CH2OH
HO
OHO
O
OH
CH2OH
OH
OH
OH
O
OH
CH2OH
HO
OH
OH
O
CH2OHHO
OH
OH
OH-gal
-gal -gal
-gal
H2O
galactose
-galactosidase (LacZ)
lactose
Other enzymes in the lac systemlactose permease (LacY)
acetylase (LacA)
lac Operator Sequence
The operator site is palindromic and lies just downstream of the transcription initiation site
lac repressor is tetrameric and blocks transcription elongation by RNAP
Induction of the lac Operon
Binding of -galactosides to the lac repressor induces a cooperative allosteric change. The inducer:lac repressor complex dissociates from the operator and transcription of the structural genes occurs.
lac Operon: Catabolite Repression
E. coli can use several sugars as carbon sources but prefers glucose because requires no energy to take it up
Glucose-sensitive operons: their expression is reduced in the presence of glucose
Catabolism of glucose inhibits expression of these operons – called catabolite repression
This mechanism allows for the lac genes to be only partially induced in the presence of lactose and glucose
Catabolite Activator ProteinPositive Control of the lac Operon
• Some promoters require an accessory protein to speed transcription
• Catabolite Activator Protein (CAP) is one such protein
• CAP is a homodimer of 22.5 kD peptides• N-term binds cAMP; C-term binds DNA• Binding of CAP-(cAMP)2 to DNA assists
formation of closed promoter complex [also called CRP (cAMP receptor protein)]
Catabolite Activator ProteinMechanism of Activation
Glucose starvation activates adenylyl cyclase increasing cAMP levels. cAMP acts as a co-inducer binding to CAP and activating transcription by binding to upstream promoter sequences.
CH2O
O
O
O P OH
O
3, 5 cyclic AMP (cAMP)
Adenine
Catabolite Activator Protein
Binding of CAP to CAP site (e.g. E. coli lac operon) induces a bend in the DNA
= cAMP
Upstream promoter sequence
CAP Activation of RNA Polymerase
The trp OperonCo-repressor Mediated Negative Control
• Encodes a leader sequence and 5 enzymes for tryptophan biosynthesis
• trp operon is always “on” unless tryptophan levels are sufficiently high to turn “off”
• Tryptophan is the co-repressor• Trp repressor binding excludes RNA polymerase from the
promoter• Trp repressor also regulates trpR and aroH operons and
is itself encoded by the trpR operon. This is autogenous regulation (autoregulation) - regulation of gene expression by the product of the gene.
trp OperonFive structural genes transcribed from the single promoter
repressor
Regulation of the trp Operon by Repression and Attenuation
Inactive Active
Attenuation The need for tryptophan is tested during translation of the “leader”
sequence (“trpL”) on all mRNA initiated at promoter.
trp expression is modulated by the tRNA-trp (“charged tRNA”) available to translate codon UGG. If the ribosomes translate these codons rapidly, transcription will stop because:
• Different segments in the leader mRNA sequence can pair with each other to form alternative stem-loop structures.
If ribosomes “stall” on the trp codons, the RNA structure formed is not a terminator. TRANSCRIPTION OF trp OPERON PROCEEDS
If ribosomes translate the leader and pass the trp codons quickly, an intrinsic terminator is formed.
TRANSCRIPTION OF trp OPERON TERMINATES
“Antiterminator”
“Terminator”
“Pause Hairpin”
Control of Gene Expression
Gene Expression
Eukaryotic Gene Transcription
Histones and Chromatin
Presence of a nucleus means transcription and translation occur in separate compartments of the cell
Larger genomes (1000X between humans and E. coli) Chromatin structure in eukaryotes limits accessibility Three RNA polymerases Eukaryote pre-mRNAs are subject to extensive post-transcriptional
modification
Eukaryotic Vs. Prokaryotic Transcription
The human genome contains approximately 1000 times the amount of DNA compared to E. coli. However, humans have only 20 times as many genes as E. coli. (98.5% of the human genome is noncoding compare to only 11% of the E. coli genome).
Genome Sizes of Various Organisms
The human genome contains 3 x 109 bp per haploid genome. If the DNA of all 46 chromosomes from one cell was linked
together, it would measure one meter in length. If the DNA in all cells of a single human was linked end to
end, it would stretch to the sun and back. How is the DNA packaged into the nucleus such that genes
are still accessible to the transcriptional regulatory proteins that control their expression?
Eukaryotic Chromatin Structure
Characterization of Human Genomic DNA
LINES - Long interspersed nuclear elements (6.1 kb full length)SINES - Short interspersed nuclear elements (300 bp)
Eukaryotic Chromatin Structure
Each eukaryotic chromosome contains a single linear supercoiled DNA molecule
Nucleoprotein material of the eukaryotic chromosome is called chromatin (complex of DNA, protein and RNA)
EM of a liver cell: cytoplasm (left) separated from the nucleus (right) by the nuclear membrane (middle)
Nuclear membrane
Nuclear pore
Heterochromatin Euchromatin
NucleusCytoplasm
Eukaryotic Chromosome Structure• Euchromatin - comprises most of
the genome, transcriptionally active, susceptible to DNaseI digestion
• Heterochromatin - highly condensed inactive chromatin located at centromeres and telomeres (less susceptible to DNase digestion)
• Centromere - attachment point for sister chromatids and spindle fibers
• Telomere - ends of chromosome
Telomeres and Telomerase
Eukaryotic Chromatin Structure DNA associates with histones proteins to produce
nucleosomes Nucleosome - the fundamental unit of organization of the
chromatin fiber Each nucleosome contains a core particle of basic proteins –
histones - which are surrounded by DNA Electron microscopy shows fiber structure of chromosomes
as “beads on a string”
Histones Small basic proteins, rich in lysine and arginine Interact with DNA through electrostatic interactions Five major types of histones:
• H1, H2A, H2B, H3 and H4• H2A, H2B, H3 and H4 form a complex of 8
proteins• DNA is supercoiled around histone octets forming
nucleosomes• H1 associates with neighboring nucleosomes to
form a more closely packed structure 30nm solenoid
(B) Structure of the histone fold.
(C) Histones 2A and 2B form a dimer through an interaction known as the handshake. Histones H3 and H4 form a dimer through the same type of interaction.
Histone Structure
Histone Structure
H2A and H2B monomers form a H2A-H2B dimer.
Similarly, H3 and H4 monomers form an H3-H4 dimer.
• H2A-H2B and H3-H4 dimers bind to form tetramers.• Two tetramers will come together to form the 8 histone octet
Histone Structure
The DNA helix makes 1.65 turns around the histone octamer.
146 base pairs of DNA wraps around each histone octet.
Histones are among the most highly conserved eukaryotic proteins. For example, the amino acid sequence of histone H4 from a pea and a cow differ at only 2 of the 102 amino acids.
DNA Wraps Around Histone Octomers
Formation of the 30nm fiber requires Histone H1 which is a globular histone that is highly conserved. Histones are positively charged and this helps to compact the negatively charged DNA.
Solenoid Formation: Role of H1 Histone
Nucleosomal Core Without DNA
Yellow: H2ARed: H2BBlue: H3Green: H4
Note ordered structure of histone amino terminal tails. These tails can contact neigh-boring nucleosomes.
When the tails are acetylated, interactions between nucleo-somes are diminished and chromatin becomes more extended or open.
Histone Tails
Solenoid
Eukaryotic Chromatin Structure
Electron micrographs of 30nm “Solenoid” structure (top) and 10nm DNA/histone “beads on a string” structure (bottom).
Eukaryotic Chromatin StructureLooping and Miniband
Eukaryotic Chromatin Structure
• Chromosomes
• Primary structure of DNA = double helix = 2nm duplex DNA
• Duplex DNA winds around histone octamers to form nucleosomes = 11 nm histone fiber
• Nucleosome fibers condense more into 30 nm chromatin fiber. Predominant form in interphase nucleus
• attachment of loops of chromatin fibers to nonhistone protein complexes = scaffold
Prokaryotes Vs. Eukaryotes