Download SF Genetics Lecture_Central Dogma_3.1 BY2208

Central Dogma
The discovery of the role of
RNA
RNA structure, synthesis and
function
!
Fundamental observations in genetics!
! Genes are located in nuclei (in eukaryotes)!
! Polypeptides are synthesised in the cytoplasm !
! Genes are made of DNA - a gene is a stretch of DNA!
! Each polypeptide is specified by one gene - a
polypeptide is a sequence of amino acids!
Cytoplasm
Nucleus
One gene one polypeptide
Two information puzzles
1!
!
!
!Transfer of information: !How can genes made of
DNA and located in the nucleus direct the !
synthesis of proteins in the cytoplasm?!
2!
!
!
!Translation of information:
!How can a !
sequence of bases (a gene) code for a sequence
of amino acids (a polypeptide)?!
!
!There is no obvious structural relationship !
between a base and an amino acid !
!
How can DNA in the Nucleus Specify the
Production of Proteins in the Cytoplams?
Is there an intermediate?
The idea that RNA acts as an intermediate was
suggested by the following findings in eukaryotic cells:
1.! DNA is mostly associated with chromosomes in the
nucleus while ribosomes that generate proteins are in the
cytoplasm.
2.! RNA is synthesised in the nucleus.
3.! RNA migrates to the cytoplasm where proteins are
synthesised.
4.! The amount of RNA is generally proportional to the amount
of protein in a cell.
One gene: one polypeptide
•! Opposite DNA strands can serve as template for
RNA
DNA makes RNA makes protein
RNA is a polymer
RNA is a polymer
Ribose (a sugar)
Usually a single strand
Bases - 4 kinds
Purines
Pyrimidines
Phosphate
Adenine
Cytosine
Guanine
Uracil
May have intramolecular
double-stranded regions
Uracil (U) like T is complementary to A
•! The four ribonucleotides found in RNA
Figure 8-2
Similarities and Differences Between DNA and RNA
RNA!
DNA!
•! Similar strand structure"
•! Can define a 5# and 3# end"
•! 2# hydroxyl in RNA: causes
stability differences)"
2’ OH
2’ H
•! Uracil in RNA takes the
place of Thymine in in DNA"
Nucleosides and nucleotides
RNA is a polymer
Sugar + Base = Nucleoside
Monomers
Sugar + Base + Phosphate = Nucleotide
Nucleoside monophosphates
AMP
GMP
CMP
UMP
AMP - adenosine monophosphate
ADP - adenosine diphosphate
ATP - adenosine triphosphate
Sugar phosphate backbone
RNA is a polymer
Monomers
Monomers
Nucleoside monophosphates
Nucleoside monophosphates
AMP
GMP
CMP
UMP
AMP
GMP
CMP
UMP
A
G
C
U
Monomer
Sugar
Base
Phosphate
Monomers
Nucleoside monophosphates
AMP
GMP
CMP
UMP
A
G
C
U
Newly discovered Classes of RNA
1)!
RNA involved in protein synthesis
1) Ribosomal RNA
(rRNA)
•! 18S, 28S and 5S rRNA
•! structural and functional components of ribosomes
•! highly abundant and stable
2) Messenger RNA
(mRNA)
•! typically about 3-500 bases long
•! encodes protein
•! multiple types, usually not abundant, unstable
3) Transfer RNA
(tRNA)
•! very small - less than 100 bases long
•! key role in translation
•! abundant and stable
Main focus of this lecture
Ribozymes
RNA molecules which have enzymatic activity
2)!
MicroRNAs
RNAs which regulate gene expression
3)!
7S RNA
Participates in transport of proteins
One gene: one polypeptide
! Synthesis of messenger RNA in
prokaryotes!
! mRNA carries the DNA code for a
polypeptide!
The Prokaryotic
Transcription Apparatus
Need to understand the following:!
•! Differences between DNA and RNA.!
•! Differences between DNA and RNA polymerases.!
Messenger RNA
•! E. coli RNA polymerase and its function.!
•! What sequences make up a prokaryotic promoter.!
•! The steps of transcription initiation and elongation.!
DNA makes mRNA makes protein:
•! The two different mechanisms of transcription
termination.!
Evidence for the Existence of mRNA
In 1956 & 1958, Volkin and colleagues undertook
studies on bacteriophage infections in E. coli.
1.! 32P-labelling of newly synthesised RNA showed it
closely resembled the base composition of phage DNA.
2.! Newly synthesised RNA is unstable.
3.! Phage RNA synthesis precedes protein synthesis.
In 1961 Spiegelman and colleagues demonstrated that
the newly synthesised RNA was complementary to the
phage DNA and not the bacterial DNA.
Base pairing explains DNA replication and RNA synthesis.
Sequence in new DNA or RNA is complementary to
the template.
In the same year, Brenner, Jacob and Meselson
demonstrated that the newly synthesised phage RNA
associates with bacterial ribosomes and the phage
proteins are synthesised on these ribosomes.
Synthesis of RNA implies existence of
an enzyme
Synthesis of RNA
1959 Samuel Weiss and others isolated an enzyme
that could synthesise RNA. Called RNA Polymerase.
To function RNAP requires:
Requirements for RNA synthesis
Double-stranded DNA as a template
DNA
4 NTPs (ATP, CTP, GTP, UTP).
ATP + GTP + CTP + UTP
Mg+
Unlike DNA polymerase, RNA polymerase does
NOT require a primer to initiate synthesis. Uses
one strand of double stranded DNA as a template.
Different Types of RNA Polymerase
RNA polymerase
RNA Synthesis is in the 5’ to 3’ Direction
DNA strand
RNA strand
Subsequent
hydrolysis of
PPi drives the
reaction forward.
OH
Similar to
DNA synthesis
ONE STRAND
IS COPIED
OH
RNA has polarity (5’ phosphate to 3’ hydroxyl)
•! Many RNAs can be simultaneously transcribed
from a gene
Terminator
Promoter
5’
Some nomenclature conventions
3’
Prokaryotic Transcription
(antisense strand)
(noncoding strand)
RNAP!
(sense strand)
(coding strand)
RNA synthesis starts at special sequences: promoters
Nomenclature!
RNA synthesis stops at special sequences: terminators
Transcription Initiation Site!
“Upstream”"
5"!
3"!
“Downstream”"
-5! -4! -3! -2! -1! +1!+2!+3!+4!+5!+6!
3"!
5"!
Direction of
transcription"
Template strand!
There is no “zero”!
What does a promoter look like?
•! Purify a short DNA molecule
•! Imagine this DNA has a promoter
•! Label double stranded DNA molecules with 32P at
on one strand on one end
•! Add RNAP to a sample (+)
•! RNAP which is big (500,000 daltons) is expected
to bind to the promoter
•! Other sample is control - no RNAP added (-)
•! Add DNAase for a short time - to cut on average
once per strand - it cuts at random at
phosphodiester bonds in the backbone of the DNA
Radioactive phosphorous
on the top strand of DNA
Radioactive phosphorous
on the top strand of DNA
RNAP!
P *!
-35!
RNAP!
-35!
-10!
RNAP!
-35!
+1!
What does a promoter look like?
+1!
RNAP “footprint” at promoter
P *!
DNAase added
-10!
Start site of transcription is +1
RNAP “footprint” at promoter
P *!
RNAP “footprint” at promoter
-10!
+1!
DNAase cuts backbone of DNA - where it is “free”
Mapping promoters
•! Note result
•! Note the ladder of DNA
fragments
•! Each 1 base shorter than
one above
•! There is a gap over several
bands
•! This shows that RNAP
protected certain bonds
from cleavage by
DNAase.
RNAP binds a region of DNA from -40 to +20"
The sequence of the non-template strand is shown"
Here are some real results
Compare the two lanes
Start
(+) RNAP added
(-) no RNAP
-10
See the two gaps in the (+) ladder
-35
Numbers are the distances in bases
from the start point of transcription
Conclude that RNAP “covers” two
regions upstream of the start site
The C lane shows the start site of
transcription (+1)
Important Promoter Features
(tested by mutations)
•! The closer the match to the consensus the stronger the
promoter (-10 and -35 boxes)
•! The absolute sequence of the spacer region (between
the -10 and -35 boxes) is not important
•! The length of the spacer sequence IS important:
TTGACA - spacer (16 to 19 base pairs) - TATAAT
-10 region!
TTGACA…16-19 bp... TATAAT"
“-35”
spacer
“-10”"
Properties of Promoters
•! Promoters typically consist of a 40 bp
region on the 5'-side of the transcription
start site
•! Two consensus sequence elements:
–! The "-35 region", with consensus TTGACA
–! The Pribnow box near -10, with consensus
TATAAT - this region is ideal for unwinding.
•! Spacers that are longer or shorter than the consensus
length make weak promoters
RNA polymerase has many functions
•!
•!
•!
•!
•!
•!
Scans DNA and identifies promoters
Binds to promoters
Initiates transcription
Elongates the RNA chain
Terminates transcription
Is responsive to regulatory proteins
(activators and repressors)
As might be expected, RNAP is a
multisubunit enzyme
Structure of RNAP in Prokaryotes
•! E.coli RNAP is a 465 kD complex
•! Sub-units: 2 !, 1 ", 1 "', 1 # (holoenzyme).
•! Core enzyme is 2 !, 1 ", 1 "’ (can transcribe but it
cannot find promoters).
•! # recognizes promoter sequences on DNA; "' binds
DNA; " binds NTPs and interacts with #.
•! ! subunits appear to be essential for assembly and for
activation of enzyme by regulatory proteins.
Assembly of RNAP: sigma factors
The assembly pathway of the core enzyme!
!
$!2
$ !2"
$ !2""’ = core enzyme!
!%$
!%%$
"$
"’!
#70$
!%$
"$
#70$
!%%$
CORE ENZYME!
Sequence-independent,!
nonspecific transcription!
initiation!
"’!
+!
#32$
#60$
vegetative!
heat shock!
nitrogen starvation!
(principal #) ! (for emergencies) ! (for emergencies) !
SIGMA SUBUNIT!
interchangeable,!
promoter recognition!
RNAP HOLOENZYME -#70!
Promoter-specific !
transcription initiation!
In the Holoenzyme:!
·! "' binds DNA !
·! " binds NTPs!
·! " and " ' together make up the active site!
·! ! subunits appear to be essential for assembly and
for activation of enzyme by regulatory proteins. They
also bind DNA.!
·! # recognizes promoter sequences on DNA!
Binding of polymerase to template
DNA
•! Polymerase binds nonspecifically to DNA with
low affinity and migrates, looking for promoter.
•! Sigma subunit recognizes promoter sequence.
•! RNA polymerase holoenzyme and promoter
form "closed promoter complex" (DNA not
unwound).
•! Polymerase unwinds about 12 base pairs to
form "open promoter complex“.
•! Transcription initiation in prokaryotes
Finding and binding
the promoter"
Closed complex
formation"
RNAP bound -40 to
+20"
Open complex
formation"
RNAP unwinds from
-10 to +2"
Binding of 1st NTP"
Requires high
purine [NTP]"
Addition of next NTPs"
Requires lower
purine [NTPs]"
Dissociation of sigma"
After RNA chain
is 6-10 NTPs long"
•! Elongation and termination of transcription
Chain Elongation
•!
•!
•!
•!
Chain Termination
Core polymerase - no sigma
Polymerase is pretty accurate - only about 1 error
in 10,000 bases (not as accurate as DNAP III)
This error rate is acceptable - many transcripts
are made from each gene
Elongation rate is 20-50 bases per second slower in G/C-rich regions and faster elsewhere
Topoisomerases precede and follow polymerase
to relieve supercoiling
Rho-Dependent Transcription Termination"
(depends on a protein AND a DNA sequence)!
Two mechanisms
G/C -rich site"
1) Rho - dependent
–! Rho, a termination factor, is an ATP-dependent
helicase
–! it moves along the RNA transcript, finds the
"bubble", unwinds it and releases the RNA
chain.
RNAP slows down"
Rho helicase
catches up"
Elongating complex is disrupted"
Chain Termination
Rho-Independent Transcription
Termination!
(depends on DNA sequence - NOT a protein factor)!
2) Rho-independent termination
-
termination sites in DNA
–! inverted repeat, rich in G:C, which forms a
stem-loop in RNA transcript
–! 6-8 A’s in DNA coding for U’s in transcript
Stem-loop structure"
Rho-independent
transcription
termination!
•! RNAP pauses when it
reaches a termination site."
•! The pause may give the
hairpin structure time to fold"
•! The fold disrupts important
interactions between the
RNAP and its RNA product"
Major Differences
between
prokaryotic and eukaryotic
transcription
•! The U-rich RNA can
dissociate from the template"
•! The complex is now
disrupted and elongation is
terminated"
Differences
between
prokaryotic and eukaryotic
transcription
Nuclear membrane
DNA is complexed with histones in nucleosomes
Promoters have many control sites for transcriptional factors
RNAP has many co-factors
Precursor to mRNA has INTRONS and EXONS
The INTRONS are spliced out in the nucleus
mRNA has a 5’ CAP structure and a 3’ polyA tail
mRNA transported from nucleus to cytoplasm
•! Prokaryotic and eukaryotic transcription and
translation compared
•! Transcription initiation in eukaryotes
•! Transcription initiation in eukaryotes
•! Transcription initiation in eukaryotes
•! Cotranscriptional processing of RNA
•! Cotranscriptional processing of RNA
•! Cotranscriptional processing of RNA
•! Cotranscriptional processing of RNA
•! Alternative splicing: alpha tropomyosin
Transcription Regulation in
Prokaryotes
•!
•!
•!
•!
Why is it necessary?
Bacterial environment changes rapidly.
Survival depends on ability to adapt.
Bacteria must make the proteins required to
survive in that environment.
•! Protein synthesis is costly (energetically).
•! Therefore, want to make enzymes when
required.
Ways to Regulate Transcription
Proteins
1. Alternate sigma factors: controls selective transcription
of entire sets of genes
vegetative
(principal)
heat shock
nitrogen
starvation
Ways to Regulate Transcription!
2. Positive Regulation (activation): a positive regulatory !
factor (activator) improves the ability of RNAP to !
bind to and initiate transcription at a weak promoter.!
s70
s32
s60
+1
TTGACA
(16-19 bp)
CNCTTGA
(13-15 bp) CCCATNT (5-9 bp) A
(5-9 bp) A
TATAAT
+1
CTGGNA (6 bp) TTGCA
Ways to Regulate Transcription!
3. Negative Regulation (repression): a negative regulatory !
factor (repressor) blocks the ability of RNAP to !
bind to and initiate transcription at a strong promoter.!
RNAP!
RNAP!
Activator!
-35!
Activator binding site!
EXAMPLE: CAP
-10!
+1
(5-9 bp) A
Repressor!
+1!
-35!
-10!
+1!
Operator!
EXAMPLE: lac REPRESSOR
Protein Synthesis is Regulated
Transcriptionally
•! Genes that encode proteins with related functions are
grouped in bacteria into transcriptional units called
“operons”!
Protein Synthesis is Regulated
Transcriptionally
Operons have three functional “parts”!
1)! structural genes: these encode proteins (usually with related
functions)"
2) Promoter"
•! This ensures that genes for enzymes in the same metabolic
pathway are all made at the same time"
3) Regulatory sequences that interact with regulatory proteins"
4) An operon may be associated with regulatory genes which
encode proteins regulating expression of that operon"
Architecture of a typical operon!
promoter"
Structural genes"
RNA transcript covers all genes in the operon"
= “polycistronic RNA”"
Operator (regulatory
sequence that binds a
repressor protein)"
By regulating a single promoter you can co-ordinate"
the expression of three genes (in this example)"