Transcript repressor protein

How cells read the genome:
From DNA to Protein
Control of Gene expression
M. Saifur Rohman, MD. PhD. FIHA. FICA
Sub topic
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From DNA to RNA
From RNA to protein
The RNA world and origin of the life
An overview of gene control
DNA binding motifs in gene regulatory
proteins
How Genetic swithes work
The molecular genetic mechanism that create
specialized cell type
Posttransciptional controls
How genome evolve
From DNA to Protein:
An overview
Protein synthesis
• DNA
• mRNA (transcription)
• Protein (translation)
From DNA to Protein
From RNA to Protein: Step by step
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The genetic code
Open reading frames
tRNA structure and production
tRNA charging - tRNA synthetases
Ribosome structure
– (components, tRNA binding, rRNA, peptide tunnel)
Peptide chain elongation
– EF-Tu, EF-G or EF1, EF2
Initiation (prokayotic & eukaryotic)
Termination
Polyribosomes
mRNA template surveillance (Quality control)
NMD, Nostop mediated decay, tmRNA
Changes in the code (selenocysteine, frameshifting, hardcoded)
Protein folding (chaperones… hsp60 & hsp70, degradation, diseases)
The RNA World
• Basic tenets of the theory
• Basic timeline
• preRNA world
• Ribozymes
• SELEX Systematic Evolution of Ligands by EXponential
enrichment
• Model of central dogma
Gene control and DNA binding motifs
• Differentiated cells contain the same DNA
• Structure of DNA binding proteins
– DNA binding and Activation domains
• Types of DNA binding motifs and how they work
• Common techniques
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Microarray
2-D gels
Gel mobility shift
DNA affinity chromatography
Footprinting
SELEX
One hybrid system
Chromatin immunoprecipitation, Chip-chip, Chip-seq
Phylogenetic footprinting
The control of gene expression
• Each cell in the human contains all the genetic
material for the growth and development of a
human
• Some of these genes will be need to be expressed all
the time
• These are the genes that are involved in of vital
biochemical processes such as respiration
• Other genes are not expressed all the time
• They are switched on an off at need
© 2007 Paul Billiet ODWS
Operons
• An operon is a group of
genes that are
transcribed at the
same time.
• They usually control an
important biochemical
process.
• They are only found in
prokaryotes.
© 2007 Paul Billiet ODWS
Jacob, Monod & Lwoff
© NobelPrize.org
The lac Operon
 The lac operon consists of three genes each
involved in processing the sugar lactose
 One of them is the gene for the enzyme βgalactosidase
 This enzyme hydrolyses lactose into glucose
and galactose
© 2007 Paul Billiet ODWS
1. When lactose is absent
• A repressor protein is continuously synthesised. It sits on a
sequence of DNA just in front of the lac operon, the Operator
site
• The repressor protein blocks the Promoter site where the
RNA polymerase settles before it starts transcribing
Repressor
protein
DNA
I
O
Regulator
gene
Operator
site
© 2007 Paul Billiet ODWS
RNA
polymerase
Blocked
z
y
lac operon
a
2. When lactose is present
• A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
• This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
DNA
I
© 2007 Paul Billiet ODWS
O
z
y
a
2. When lactose is present
• A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
• This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
DNA
I
© 2007 Paul Billiet ODWS
O
z
y
Promotor site
a
3. When both glucose and lactose are
present
• This explains how the lac operon is
transcribed only when lactose is present.
• BUT….. this does not explain why the operon
is not transcribed when both glucose and
lactose are present.
© 2007 Paul Billiet ODWS
• When glucose and lactose are present RNA
polymerase can sit on the promoter site but it is
unstable and it keeps falling off
Repressor protein
removed
RNA polymerase
DNA
I
O
z
y
Promotor site
a
4. When glucose is absent and lactose
is present
• Another protein is needed, an activator protein. This
stabilises RNA polymerase.
• The activator protein only works when glucose is absent
• In this way E. coli only makes enzymes to metabolise other
sugars in the absence of glucose
Activator
protein steadies
the RNA
polymerase
Transcription
DNA
I
O
z
y
Promotor site
© 2007 Paul Billiet ODWS
a
Carbohydrates Activator
protein
Repressor
protein
RNA
polymerase
lac Operon
+ GLUCOSE
+ LACTOSE
Not bound
to DNA
Lifted off
operator site
Keeps falling
off promoter
site
No
transcription
+ GLUCOSE
- LACTOSE
Not bound
to DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
- LACTOSE
Bound to
DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
+ LACTOSE
Bound to
DNA
Lifted off
Sits on the
operator site promoter site
Transcription
© 2007 Paul Billiet ODWS
• Control of Gene Expression
• 1. DNA-Protein Interaction
• 2. Transcription Regulation
• 3. Post-transcriptional Regulation
Neuron and lymphocyte
Different morphology, same genome
Six Steps at which eucaryotic gene expression are controlled
Regulation at DNA levels
Double helix Structure
The outer surface difference of base pairs without opening
the double helix
Hydrogen bond donor:
blue
Hydrogen bond
acceptor: red
Hydrogen bond: pink
Methyl group: yellow
DNA recognition code
One typical contact of Protein
and DNA interface
In general, many of them will
form between a protein and a
DNA
DNA-Protein Interaction
1.
2.
3.
Different protein motifs binding to DNA: Helix-turn-Helix
motif; the homeodomain; leucine zipper; helix-loop-helix;
zinc finger
Dimerization approach
Biotechnology to identify protein and DNA sequence
interacting each other.
Helix-turn-Helix
C-terminal binds to major groove, N-terminal helps to position
the complex, discovered in Bacteria
Homeodomain Protein in Drosophila utilizing
helix-turn-helix motif
Zinc Finger Motifs
Utilizing a zinc in the center
An alpha helix and two beta sheet
An Example protein (a mouse DNA
regulatory protein) utilizing Zinc
Finger Motif
Three Zinc Finger Motifs forming the
recognition site
A dimer of the zinc finger domain of the glucocorticoid receptor (belonging to intracellular
receptor family) bound to its specific DNA sequence
Zinc atoms stabilizing DNA-binding Helix and dimerization interface
Beta sheets can also recognize DNA sequence
(bacterial met repressor binding to s-adenosyl methionine)
Leucine Zipper Dimer
Same motif mediating both DNA
binding and Protein dimerization
(yeast Gcn4 protein)
Homodimers and heterodimers can recognize
different patterns
Helix-loop-Helix (HLH) Motif and its dimer
Truncation of HLH tail (DNA binding domain) inhibits binding
Six Zinc Finger motifs and their interaction with DNA
Gel-mobility shift assay
Can identify the sizes of proteins
associated with the desired DNA
fragment
DNA affinity Chromatography
After obtain the protein, run mass spec, identify aa sequence, check genome, find
gene sequence
Assay to determine the gene
sequence recognized by a
specific protein
Chromatin Immunoprecipitation
In vivo genes bound to a known protein
Summary
• Helix-turn-Helix, homeodomain, leucine
zipper, helix-loop-helix, zinc-finger motif
• Homodimer and heterodimer
• Techniques to identify gene sequences bound
to a known protein (DNA affinity
chromatography) or proteins bound to known
sequences (gel mobility shift)
Gene Expression Regulation
Transcription
Tryptophan Gene Regulation (Negative control)
Operon: genes adjacent to each other and are transcribed from a single promoter
Different Mechanisms of
Gene Regulation
The binding site of
Lambda Repressor
determines its
function
Act as both activator
and repressor
Combinatory Regulation of Lac Operon
CAP: catabolite activator protein; breakdown of lactose when glucose is low and lactose is present
The difference of Regulatory
system in eucaryotes and bacteria
1.
2.
3.
Enhancers from far distance over promoter regions
Transcription factors
Chromatin structure
Gene Activation at a distance
Regulation of an eucaryotic gene
TFs are similar, gene regulatory proteins
could be very different for different gene
regulations
Functional Domain
of gene activation
protein
1. Activation
domain and 2. DNA
binding domain
Gene Activation by
the recruitment of
RNA polymerase II
holoenzyme
Gene engineering revealed the function of gene
activation protein
Directly fuse the mediator protein to enhancer
binding domain, omitting activator domain, similar
enhancement is observed
Gene regulatory proteins help the recruitment and
assembly of transcription machinery
(General model)
Gene activator proteins recruit
Chromatin modulation proteins
to induce transcription
Two mechanisms of histone
acetylation in gene
regulation
a. Histone acetylation further
attract activator proteins
b. Histone acetylation
directly attract TFs
Synergistic Regulation
Transcription synergy
5 major ways of
gene repressor
protein to be
functional
Protein Assembled to form commplex to Regulate
Gene Expression
Integration for Gene Regulation
Regulation of Gene Activation Proteins
Insulator Elements (boundary elements) help to
coordinate the regulation
Gene regulatory proteins
can affect transcription
process at different steps
The order of process may
be different for different
genes
Summary
• Gene activation or repression proteins
• DNA as a spacer and distant regulation
• Chromatin modulation, TF assembly,
polymerase recruitment
• combinatory regulations
Genetic Switches
Positive, negative and combinatorial control of transcription in
bacteria
Trp and Lac operons
Lambda repressor
DNA bending and protein-protein interactions on DNA
Differences in transcription regulation between prokaryotes and
eukaryotes
The structure of a eukaryotic gene control region
How eukaryotic transcriptional activators work
How eukaryotic transcriptional repressors work
Steps of eukaryotic transcriptional activation
Transcription factor complexes, coactivators and corepressors
synergy
Control of Drosophila even-skipped (eve)
Locus control regions and insulators
Creating Specialized Cells
Phase variation in Salmonella
Yeast mating type switching
Regulation of lambda phage lysogeny: flip-flop
Four types of feedback
Positive and negative transcription feedback loops
Examples:
Circadian clocks: (don’t need to know details)
Myogenic proteins and muscle cell formation
Eye development in Drosophila
Creation of cell types by a few transcription factors
Mechanisms by which patterns of gene expression
can be passed to daughter cells:
X-inactivation
Cytosine methylation
Genomic imprinting
CpG islands
Post transcriptional controls
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Post-initiation transcriptional control of gene expression
• attenuation
Alternative splicing
• Regulation of alternative splicing
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Transcript cleavage
• Secreted verses membrane bound antibodies
RNA editing especially as it related to human cells
RNA transport and localization
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Negative control of translation initiation
• Export of HIV RNAs from the nucleus
• Localization in the cytoplasm
• Bacteria (ex. Bacterial ribosomal proteins)
• How do translational repressor work in eukaryotes
–Aconitase
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• Phosphorylation of eIF-2
• uORFs
IRES
Control of mRNA stability
RNA interference, miRNAs, siRNAs
Transcription
• The transcription cycle
• The structure of E. coli RNA polymerase
• Sigma70 promoter structure (-10 region & variants)
– Sigma factors
• Subunits of bacterial RNA polymerase
• Two types of terminators
• Eukaryotic RNA pols
– General composition of the polymerases
– General transcriptions factors
– TATA and other promoter DNA sequence signals
– Mediator complex
• Elongation
• RNA capping, Splicing, Cleavage and polyAdenylation
• Differences between prokaryotic and eukaryotic transcription
Splicing
• Spliceosome structure and mechanism of splicing
• Different types of splicing (3 major types)
• Group I and II introns
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1. Transcription
2. RNA Modification and Splicing
3. RNA transportation
4. Translation
Processing of eukaryotic pre-mRNA: the
classical texbook picture
Alternative picture: co-transcriptional
pre-mRNA processing
• This picture is more
realistic than the
previous one,
particularly for long
pre-mRNAs
Heterogenous ribonucleoprotein patricles
(hnRNP) proteins
• In nucleus nascent RNA transcripts are associated
with abundant set of proteins
• hnRNPs prevent formation of secondary
structures within pre-mRNAs
• hnRNP proteins are multidomain with one or
more RNA binding domains and at least one
domain for interaction with other proteins
• some hnRNPs contribute to pre-mRNA
recognition by RNA processing enzymes
• The two most common RNA binding domains are
RNA recognition motifs (RRMs) and RGG box (five
Arg-Gly-Gly repeats interspersed with aromatic
residues)
3D structures of RNA recognition
motif (RRM ) domains
Capping
p-p-p-N-p-N-p-N-p….
Capping enzyme
(mCE)
p-p-N-p-N-p-N-p…
GMP
mCE (another subunit)
G-p-p-p-N-p-N-p-N-p…
S-adenosyl
methionine
methyltransferases
CH3
G-p-p-p-N-p-N-p-N-p…
CH3 CH3
The capping enzyme
• A bifunctional enzyme with both 5’-triphosphotase
and guanyltransferase activities
• In yeast the capping enzyme is a heterodimer
• In metazoans the capping enzyme is monomeric
with two catalytic domains
• The capping enzyme specific only for RNAs,
transcribed by RNA Pol II (why?)
Capping mechanism in mammals
Growing RNA
DNA
Capping enzyme is allosterically controlled by CTD domains
of RNA Pol II and another stimulatory factor hSpt5
Polyadenylation
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Poly(A) signal recognition
Cleavage at Poly(A) site
Slow polyadenylation
Rapid polyadenylation
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G/U: G/U or U rich region
CPSF: cleavage and
polyadenylation specificity
factor
CStF: cleavage stimulatory
factor
CFI: cleavage factor I
CFII: cleavage factor II
PAP: Poly(A) polymerase
PAP
CPSF
PABPII- poly(A) binding protein II
PABP II functions:
1.
rapid polyadenylation
2.
polyadenylation termination
Link between polyadenylation and
transcription
FCP1 Phosphatase
removes phospates from
CTDs
Pol II gets
recycled
mRNA
Pol II
aataaa
c
t
d p
p
PolyA – binding
factors
cap
degradation
p
p
cap
polyA
mRNA gets cleaved and
polyadenylated
cap
splicing,nu
clear
transport
Splicing
The size distribution of exons and introns in
human, Drosophila and C. elegans genomes
Consensus sequences around the
splice site
YYYY
Molecular
mechanism
of splicing
Small nuclear RNAs U1-U6
participate in splicing
• snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins
each, forming small nuclear ribonucleoprotein particles (snRNPs)
• Sm- binding sites for snRNP proteins
Additional factors of exon recognition
ESE - exon splicing enhancer sequences
SR – ESE binding proteins
U2AF65/35 – subunits of U2AF factor, binding to pyrimidine-rich regions and 3’
splice site
The essential steps in splicing
Binding of U1 and U2
snRNPs
Binding of U4, U5
and U6 snRNPs
Rearrangement of
base-pair interactions
between snRNAs,
release of U1 and U4
snRNPs
The catalytic core,
formed by U2 and U6
snRNPs catalyzes the
first transesterification
reaction
Further rearrangements
between U2, U6 and U5
lead to second
transesterification
reaction
The spliced lariat is linearized by debranching enzyme and
further degraded in exosomes
Not all intrones are completely degraded. Some end up as
functional RNAs, different from mRNA
Co-transciptional splicing
mRNA
Pol II
c
t
d
SRs
snRNPs
p
p
SCAFs: SR- like CTD
– associated factors
Intron
cap
Self-splicing introns
• Under certain nonphysiological conditions in
vitro, some introns can get spliced without aid
of any proteins or other RNAs
• Group I self-splicing introns occur in rRNA
genes of protozoans
• Group II self-splicing introns occur in
chloroplasts and mitochondria of plants and
fungi
Group I introns utilize guanosine cofactor, which is not part of RNA chain
Comparison of secondary structures of group II selfsplicing introns and snRNAs
Spliceosome
• Spliceosome contains snRNAs, snRNPs and many
other proteins, totally about 300 subunits.
• This makes it the most complicted macromolecular
machine known to date.
• But why is spliceosome so extremely complicated if
it only catalyzes such a straightforward reaction as
an intron deletion? Even more, it seems that some
introns are capable to excise themselves without
aid of any protein, so why have all those 300
subunits?
• No one knows for sure, but there might be at
least 4 reasons:
• 1. Defective mRNAs cause a lot of problems for
cells, so some subunits might assure correct
splicing and error correction
• 2. Splicing is coupled to nuclear transport, this
requires accessory proteins
• 3. Splicing is coupled to transcription and this
might require more additional accessory proteins
• 4. Many genes can be spliced in several
alternative ways, which also might require
additional factors
One gene – several proteins
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Cleavage at alternative poly(A) sites
Alternative promoters
Alternative splicing of different exons
RNA editing
Alternative splicing, promoters &
poly-A cleavage
RNA editing
• Enzymatic altering of pre-mRNA sequence
• Common in mitochondria of protozoans and plants and
chloroplasts, where more than 50% of bases can be
altered
• Much rarer in higher eukaryotes
Editing of human apoB pre-mRNA
The two types of editing
1) Substitution editing
• Chemical altering of individual nucleotides
• Examples: Deamination of C to U or A to I
(inosine, read as G by ribosome)
2) Insertion/deletion editing
•Deletion/insertion of nucleotides (mostly uridines)
•For this process, special guide RNAs (gRNAs) are
required
Guide RNAs (gRNAs) are required for editing
Organization of pre-rRNA genes
in eukaryotes
Electron micrograph of tandem
pre-rRNA genes
Small nucleolar RNAs
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~150 different nucleolus restricted RNA species
snoRNAs are associated with proteins, forming small
nucleolar ribonucleoprotein particles (snoRNPs)
• The main three classes of snoRNPs are envolved in
following processes:
a) removing introns from pre-rRNA
b) methylation of 2’ OH groups at specific sites
c) converting of uridine to pseudouridine
What is this pseudouridine good for?
Uridine ( U )
Pseudouridine ( Y )
• Pseudouridine Y is found in RNAs that have a tertiary
structure that is important for their function, like rRNAs,
tRNAs, snRNAs and snoRNAs
• The main role of Y and other modifications appears to be
the maintenance of three-dimensional structural integrity
in RNAs
Where do snoRNAs come from?
• Some are produced from their own promoters by
RNA pol II or III
• The majority of snoRNAs come from introns of
genes, which encode proteins involved in
ribosome synthesis or translation
• Some snoRNAs come from intrones of genes,
which encode nonfuctional mRNAs
Assembly of
ribosomes
Processing of pre-tRNAs
RNase P
cleavage site
Splicing of pre-tRNAs is different
from pre-mRNAs and pre-rRNAs
• The splicing of pre-tRNAs is catalyzed by
protein only
• A pre-tRNA intron is excised in one step,
not by two transesterification reactions
• Hydrolysis of GTP and ATP is required to
join the two RNA halves
Macromolecular transport across the
nuclear envelope
The central channel
• Small metabolites, ions and globular
proteins up to ~60 kDa can diffuse freely
through the channel
• Large proteins and ribonucleoprotein
complexes (including mRNAs) are
selectively transported with the assistance
of transporter proteins
Proteins which are transported into nucleus contain
nuclear location sequences
Two different kinds of nuclear location sequences
basic
hydrophobic
importin a
importin b
importin b
nuclear import
Artifical fusion of a nuclear localization signal to a
cytoplasmatic protein causes its import to nucleus
Mechanism for nuclear “import”
Mechanism for nuclear “export”
Mechanism for mRNA transport to cytoplasm
Example of regulation at nuclear transport level:
HIV mRNAs
After mRNA reaches the cytoplasm...
• mRNA exporter, mRNP proteins, nuclear capbinding complex and nuclear poly-A binding
proteins dissociate from mRNA and gets back to
nucleus
• 5’ cap binds to translation factor eIF4E
• Cytoplasmic poly-A binding protein (PABPI) binds
to poly-A tail
• Translation factor eIF4G binds to both eIF4E and
PABPI, thus linking together 5’ and 3’ ends of
mRNA
Quality control of translation
in bacteria
Rescue the incomplete
mRNA process and add
labels for proteases
Folding of the proteins
Is required before functional
Folding process starts at ribosome
Protein Folding Pathway
Molecular Chaperone
An example of molecular chaperone functions
Hsp70, early binding to proteins after synthesis
An example of molecular chaperone functions (chaperonin)
Hsp60-like protein, late
The Fate of Proteins after translation
E1: ubiquitin activating enzyme; E2/3: ubiquitin ligase
The production of proteins
• RNA translation (Protein synthesis), tRNA,
ribosome, start codon, stop codon
• Protein folding, molecular chaperones
• Proteasomes, ubiquitin, ubiqutin ligase
How Genomes evolve
• Mutations, gene deletions, chromosomal rearrangements,
transposable elements, horizontal transfer, inversions, gene
duplication, whole genome duplication
• Phylogenetic trees
• Sequence alignments
• Chromosomal rearrangements
• Gene duplication
• Neofunctionalization, subfunctionalization,
• Whole genome duplication
• SNPs (mutations within a genome)
• Haplotypes
• CNVs
How genome evolve
• No Evolution !!!
Thank You