Transcript No Slide Title

Gene Expression:
Nucleus
DNA
Pol II transcription
Primary RNA transcript
Multiple, Spatially and
Temporally Distinct Steps
Carried out by Distinct
Cellular Machinery
Nuclear processing
Capping
Splicing
Polyadenylation
Cytoplasm
Mature
mRNA
Mature mRNA
Export
Degradation
Protein
Translation
- Regulation Can Be at Several Different Levels
- Dynamic Protein Association
How can the cell distinguish between
(1) intron-containing pre-mRNAs
(2) spliced mRNAs
(3) intronless mRNAs
to ensure that (1) are retained in the nucleus while (2 and 3) are exported to
the cytoplasm?
Mechanisms of mRNA turnover
(NMD, mRNAs contain a premature stop codon, an in-frame stop codon
within a retained intron, or an extended 3’ UTR due to improper polyadenylation site usage)
(mRNAs lack an in-frame termination codon-decay occurs in the cytoplasm)
Nuclear retention of unspliced mRNAs
•An intact 5’ splice site and branchpoint are
required for nuclear retention of pre-mRNAs
•Numerous splicing factors, including U1
snRNA and branchpoint binding protein
(BBP/SF1), have been found to affect nuclear
retention of pre-mRNAs
•In yeast, perinuclearly located Mlp1 physically
retains improperly spliced pre-mRNAs but
does not affect the splicing process itself
Thus, it appears that Mlp1 retains pre-mRNAs
that assemble into a spliceosome but fail to
proceed through splicing before reaching the
nuclear pore complex
green-Mlp1 detected with an antibody
red-the nucleolar protein Nop1 detected
with an antibody
Galy, V. et al. Cell 116, 63-73 (2004)
Coupling of Transcriptional and
Post-Transcriptional Events
mRNA Surveillance
RNA-Mediated Gene Silencing
Coupling of Transcriptional
and Post-Transcriptional Events
A) CTD of RNA Pol II
- binding platform for mRNA
processing components
B) TREX
- couples TRanscription and EXport
C) Exon Junction Complex (EJC)
- splicing mark and coupler
mRNA Surveillance
A) Quality Control Mechanism
- ‘Process vs. Discard’
B) Nonsense Mediated Decay
- Elimination of mRNAs with
Premature Stop Codons (PTCs)
RNA-Mediated Gene Silencing
A) Post-Transcriptional Gene Silencing
(PTGS or RNA Interference)
- mRNA degradation
- translation block
B) Transcriptional Gene Silencing (TGS)
- DNA methylation
- Heterochromatin formation
- DNA rearrangement/elimination
Gene Expression:
Nucleus
Linear Assembly Line?
DNA
Pol II transcription
Primary RNA transcript
Nuclear processing
Capping
Splicing
Polyadenylation
Cytoplasm
Mature
mRNA
Mature mRNA
Export
Degradation
Protein
Translation
Gene Expression:
Complex Network of Coupled Interactions
Maniatis, T., and Reed , R. (2002 ). Nature 416, 499-506.
Co-Transcriptional Recruitment of
pre-mRNA Processing Factors
TREX
5’
Nascent mRNA
CTD
5’ Capping Enzymes
o Splicing Machinery
o 3’ Cleavage/PolyA Factors
o Export Receptors
o
Jensen , T.H., Dowe r, K., Libri, D., and Ro sbash , M. (2003 ). Mol Cell 11, 1129 -1138.
Transcription
Export
T-REX
Transcription Elongation Factors
(THO Complex: Hpr1p, Tho2p, Mft1p, Thp2p)
Export Factors (Yra, Sub2)
Tex1 (unknown function)
http://home.wxs.nl/~vrie0388/trex.JPG
Nuclear mRNA Surveillance and Quality Control:
Process or Discard
Degradation by
Nuclear Exosome
(3’-5’ exonucleases)
Transcriptional
Coupling & mRNA
Surveillance
RNA
Pol II
Nascent
mRNA
NPC
Exosome
Jensen , T.H., and Rosb ash, M.
(2003) . Nat Struct Biol 10, 10-12.
Ribosome
Exon Junction Complex
The Splicing Process Leaves its ‘Mark’
EJC
• The EJC assembles
following splicing.
• EJC is deposited 20-24 nts
upstream of the exon-exon
junction.
• EJC as a ‘Molecular Link’
between splicing and
downstream events.
EJC: A Molecular Link Between mRNA Splicing and
Subsequent Events (Export, Localization, Decay, Translation, etc.)
Tange, T.O., Nott, A., and Moo re, M.J. (2004). Curr Opin Cell Biol 16, 279-284.
RNA-Mediated Gene Silencing
RNA Interference (PTGS)
Transcriptional Gene Silencing (TGS)
Common Trigger:
Mechanism of RNAi: Gene Silencing
directed by ~22nt RNAs
dsRNA
DICER
processing
~22nt siRNAs
RISC
target
mRNA
recognition
(Argonaute)
degradation
RISC: RNA-Induced Silencing Complex (contains effector nuclease)
MicroRNAs and SiRNAs: Small but Mighty ‘Riboregulators’!
- Viruses
- Transposons
- Repeat Elements
- Exogenous
Endogenous
Host genes
MicroRNA
Precursor
Translational
Repression
mRNA
Degradation
Proposed Biologic Roles
‘Immune System’ of the Genome
• Antiviral Defense
• Suppress Transposon Activity
• Gene Regulation (Silencing)
(e.g. MicroRNAs, Heterochromatin)
RNA-Mediated Gene Silencing
RITS
RNA-Induced Initiator of
Transcriptional Gene Silencing
(siRNAs, Ago1, Chp1 (chromodomain protein), Tas3)
RITS
RITS
DNA methylation
- Multiple dsRNA Inputs
- Different Silencing Events
Bartel, D.P. (2004 ). Cell 116, 281-297.
Mechanism of RNAi-Mediated Heterochromatin Formation
Trigger: Repetitive DNA
RITS
DNA
2. Recruitment
Two Very Different Outcomes!
1. Histone 3
Methylation
TRANSLATION
Molecular Biology
Familiarity with basic concepts is assumed, including:
 nature of the genetic code
 maintenance of genes through DNA replication
 transcription of information from DNA to mRNA
 translation of mRNA into protein.
DNA
mRNA
protein
Genetic code
The genetic code is based on the sequence of bases along
a nucleic acid.
Each codon, a sequence of 3 bases in mRNA, codes for
a particular amino acid, or for chain termination.
Some amino acids are specified by 2 or more codons.
Synonyms (multiple codons for the same amino acid) in
most cases differ only in the 3rd base. Similar codons
tend to code for similar amino acids. Thus effects of
mutation are minimized.
Genetic Code
1st base
U
UUU Phe
U
UUC Phe
UUA Leu
UUG Leu
CUU Leu
C
CUC Leu
CUA Leu
CUG Leu
AUU Ile
A
AUC Ile
AUA Ile
AUG Met*
GUU Val
G
GUC Val
GUA Val
GUG Val
*Met and initiation.
2nd base
C
UCU Ser
UCC Ser
UCA Ser
UCG Ser
CCU Pro
CCC Pro
CCA Pro
CCG Pro
ACU Thr
ACC Thr
ACA Thr
ACG Thr
GCU Ala
GCC Ala
GCA Ala
GCG Ala
A
UAU Tyr
UAC Tyr
UAA Stop
UAG Stop
CAU His
CAC His
CAA Gln
CAG Gln
AAU Asn
AAC Asn
AAA Lys
AAG Lys
GAU Asp
GAC Asp
GAA Glu
GAG Glu
3rd base
G
UGU Cys
UGC Cys
UGA Stop
UGG Trp
CGU Arg
CGC Arg
CGA Arg
CGG Arg
AGU Ser
AGC Ser
AGA Arg
AGG Arg
GGU Gly
GGC Gly
GGA Gly
GGG Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Prokaryotic genes
Prokaryotes (intronless protein coding genes)
Upstream (5’)
promoter
TAC
Gene region
Downstream (3’)
DNA
Transcription (gene is encoded on minus strand ..
And the reverse complement is read into mRNA)
ATG
5´ UTR CoDing Sequence (CDS)
mRNA
3´ UTR
ATG
Translation: tRNA read off each codons, 3 bases
at a time, starting at start codon until it reaches a
STOP codon.
protein
Prokaryotic genes (operons)
Prokaryotes (operon structure)
upstream promoter
downstream
Gene 1
Gene 2
Gene 3
In prokaryotes, sometimes genes that are part of the same
operational pathway are grouped together under a single
promoter. They then produce a pre-mRNA which
eventually produces 3 separates mRNA´s.
Bacterial Gene Structure of signals
- translation binding site (shine-dalgarno) 10 bp upstream of AUG
(AGGAGG)
- One or more Open Reading Frame
•start-codon (unless sequence is partial)
•until next in-frame stop codon on that strand ..
Separated by intercistronic sequences.
- Termination
Eukaryotic Central Dogma
In Eukaryotes ( cells where the DNA is sequestered in a separate nucleus)
The DNA does not contain a duplicate of the coding gene, rather exons must be spliced. (
many eukaryotes genes contain no introns! .. Particularly true in ´lower´ organisms)
mRNA – (messenger RNA) Contains the assembled copy of the gene. The mRNA acts as a
messenger to carry the information stored in the DNA in the nucleus to the cytoplasm
where the ribosomes can make it into protein.
tRNA
The genetic code is read during translation via adapter
molecules, tRNAs, that have 3-base anticodons
complementary to codons in mRNA.
"Wobble" during reading of the mRNA allows some
tRNAs to read multiple codons that differ only in the
3rd base.
There are 61 codons specifying 20 amino acids.
Minimally 31 tRNAs are required for translation, not
counting the tRNA that codes for chain initiation.
Mammalian cells produce more than 150 tRNAs.
tRNA ( transfer RNA)
is a small RNA that has a very specific secondary and tertiary structure such that it can
bind an amino acid at one end, and mRNA at the other end. It acts as an adaptor to carry
the amino acid elements of a protein to the appropriate place as coded for by the mRNA. T
Secondary structure of tRNA
Threedimensional
Tertiary
structure
RNA structure:
Most RNAs have
secondary structure,
consisting of stem &
loop domains.
A
:
U
U
:
A
A
:
U
stem
C
:
G
C UG
C
U
:
G
U
C U
loop
Double helical stems arise from base pairing between
complementary stretches of bases within the same strand.
Loops occur where lack of complementarity, or the
presence of modified bases, prevents base pairing.
anticodon loop
The “cloverleaf” model of
tRNA secondary structure
emphasizes the 2 major types
of secondary structure,
stem and loop domains.
tRNA
acceptor
stem
tRNAs typically include many modified bases,
particularly in the loop domains.
Tertiary structure depends on interactions of bases at
more distant sites. Many of these interactions involve
non-standard base pairing and/or interactions involving
three or more bases.
tRNAs usually fold into an L-shaped tertiary structure.
anticodon loop
tRNA
Extending out from the
"acceptor stem", the 3' end of
every tRNA has the sequence CCA.
acceptor
stem
The appropriate amino
acid is attached to the
ribose of the terminal A
(in red) at the 3' end.
The anticodon loop is
at the opposite end of
the L shape.
anticodon
Phe
tRNA
acceptor
stem
Tertiary base pairs
#46
(m7G)
#22
G
Tertiary base
Phe
pairs in tRNA
#13
C
#46
(m7G)
#22
G
#13
C
Tertiary base
pairs in tRNAPhe
Non-standard H bond interactions, some linking 3 bases,
help stabilize the L-shaped tertiary structure of tRNA. This
example is from NDB file 1TN2. H atoms are not shown.
O
R
H
C
C
O
O

O
P
Amino acid
P
C
O
O
P
O
O
CH2
O
H
ATP

O
R
O
O
NH3+
H
C
O
Adenine
O
H
H
OH
H
OH
O
O
NH2
Aminoacyl-AMP
P
O
CH2
O
H
Adenine
O
H
H
OH
H
OH
 PPi
Aminoacyl-tRNA Synthetases catalyze linkage of the
appropriate amino acid to each tRNA. The reaction occurs
in two steps.
In step 1, an O atom of the amino acid a-carboxyl attacks
the P atom of the initial phosphate of ATP.
O
R
H
C
C
O
O
P
O
CH2
O
NH2
H
Aminoacyl-AMP
In step 2, the
2' or 3' OH of
the terminal
adenosine of
tRNA attacks
the amino acid
carbonyl C
atom.
O
H
H
OH
H
OH
tRNA
AMP

tRNA
Adenine
O
O
P
O
CH2
O
Adenine
O
H
H
H
3’
2’
H
OH
O
C
O
HC
R
NH3+
(terminal 3’nucleotide
of appropriate tRNA)
Aminoacyl-tRNA
Aminoacyl-tRNA Synthetase
Summary of the 2-step reaction:
1. amino acid + ATP  aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA  aminoacyl-tRNA + AMP
The 2-step reaction is spontaneous overall, because the
concentration of PPi is kept low by its hydrolysis,
catalyzed by Pyrophosphatase.
There is a different Aminoacyl-tRNA Synthetase
(aaRS) for each amino acid.
Each aaRS recognizes its particular amino acid and the
tRNAs coding for that amino acid.
Accurate translation of the genetic code depends on
attachment of each amino acid to an appropriate tRNA.
Domains of tRNA recognized by an
aaRS are called identity elements.
Most identity elements are in the
anticodon loop
acceptor stem & anticodon loop.
Aminoacyl-tRNA Synthetases arose
early in evolution. The earliest
aaRSs probably recognized tRNAs
acceptor
stem
only by their acceptor stems.
tRNA
tRNA
O
O
P
O
(terminal 3’nucleotide
of appropriate tRNA)
O
O
H
H
There are 2 families
of Aminoacyl-tRNA
Synthetases:
Class I & Class II.
Adenine
CH2
O
H
3’
2’
H
OH
C
O
HC
R
NH3+
Aminoacyl-tRNA
Two different ancestral proteins evolved into the 2 classes
of aaRS enzymes, which differ in the architecture of their
active site domains. They bind to opposite sides of the
tRNA acceptor stem, resulting in aminoacylation of a
different OH of the tRNA (2' or 3').
Class I aaRSs:
Identity elements usually include residues of the
anticodon loop & acceptor stem.
Class I aaRSs aminoacylate the 2'-OH of adenosine at
their 3' end.
Class II aaRSs:
Identity elements for some Class II enzymes do not
include the anticodon domain.
Class II aaRSs tend to aminoacylate the 3'-OH of
adenosine at their 3' end.
Proofreading/quality control:
Some Aminoacyl-tRNA Synthetases are known to have
separate catalytic sites that release by hydrolysis
inappropriate amino acids that are misacylated or mistransferred to tRNA.
E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a
small percentage of the time activates the closely related
amino acid valine to valine-AMP.
After valine is transferred to tRNAIle, to form Val-tRNAIle,
it is removed by hydrolysis at a separate active site of
IleRS that accommodates Val but not the larger Ile.
In some bacteria, editing of some misacylated tRNAs is
carried out by separate proteins that may be evolutionary
precursors to editing domains of aa-tRNA Synthetases.
Some amino acids are modified after being linked to tRNA.
 E.g., in prokaryotes & in mitochondria the initiator
tRNAfMet is first charged with methionine.
Methionyl-tRNA formyltransferase then catalyzes
formylation of the methionine moiety, using THF as
formyl donor, to yield fMet-tRNAfMet.
 In some prokaryotes, a non-discriminating aaRS
loads aspartate onto tRNAAsn.
The aspartate moiety is then converted by an amidotransferase to asparagine, yielding Asn-tRNAAsn.
Glu-tRNAGln is similarly formed and converted to GlntRNAGln in such organisms.
RIBOSOMES
Ribosome Composition (S = sedimentation coefficient)
Ribosome
Source
E. coli
Whole
Ribosome
70S
Small
Subunit
30S
16S RNA
21 proteins
Rat
cytoplasm
80S
40S
18S RNA
33 proteins
Large
Subunit
50S
23S & 5S
RNAs
31 proteins
60S
28S, 5.8S, &5S
RNAs
49 proteins
Eukaryotic cytoplasmic ribosomes are larger and more
complex than prokaryotic ribosomes. Mitochondrial and
chloroplast ribosomes differ from both examples shown.
5S rRNA
“crown” view
displayed as
ribbons & sticks.
PDB 1FFK
Structures of large & small subunits of bacterial &
eukaryotic ribosomes have been determined, by X-ray
crystallography & by cryo-EM with image reconstruction.
Consistent with predicted base pairing, X-ray crystal
structures indicate that ribosomal RNAs (rRNAs) have
extensive secondary structure.
Structure of the E. coli Ribosome
large subunit
tRNA
EF-G
small subunit
mRNA
location
The cutaway view at right shows positions of tRNA (P, E
sites) & mRNA (as orange beads). EF-G will be discussed
later. This figure was provided by Joachim Frank, whose
lab at the Wadsworth Center carried out the cryo-EM and
3D image reconstruction on which the images are based.
Small Ribosomal Subunit
 In the translation complex, mRNA threads through a
tunnel in the small ribosomal subunit.
 tRNA binding sites are in a cleft in the small subunit.
 The 3' end of the 16S rRNA of the bacterial small
subunit is involved in mRNA binding.
 The small ribosomal subunit is relatively flexible,
assuming different conformations.
E.g., the 30S subunit of a bacterial ribosome was
found to undergo specific conformational changes
when interacting with a translation initiation factor.
Small
ribosomal
subunit of a
thermophilic
bacterium:
rRNA in
monochrome;
proteins in
varied colors.
30S ribosomal subunit
spacefill display
PDB 1FJF
ribbons
The overall shape of the 30S ribosomal subunit is largely
determined by the rRNA. The rRNA mainly consists of
double helices (stems) connected by single-stranded loops.
The proteins generally have globular domains, as well as
long extensions that interact with rRNA and may stabilize
interactions between RNA helices.
Large ribosome subunit:
The interior of the large
subunit is mostly RNA.
Proteins are distributed
mainly on the surface.
PDB 1FFK
Large
Ribosome
Subunit
Some proteins have long
tails that extend into the
interior of the complex.
These tails, which are
highly basic, interact
with the negatively
charged RNA.
"Crown" view with RNAs blue, in
spacefill; proteins red, as backbone.
The active site domain
for peptide bond
formation is essentially
devoid of protein.
PDB 1FFK
Large
Ribosome
Subunit
Peptidyl transferase is
attributed to 23S rRNA,
making this RNA a
"ribozyme."
A universally conserved
adenosine base serves as
a general acid base
during peptide bond
formation.
"Crown" view with RNAs blue, in
spacefill; proteins red, as backbone.
Protein synthesis takes PDB 1FFK
place in a cavity within
the ribosome.
Nascent polypeptides
emerge through a
tunnel in the large
subunit.
Some nascent proteins
then pass through a
channel into the ER
lumen, or across the
cytoplasmic membrane Large ribosome subunit.
Backbone display with RNAs blue. View
and out of the cell in
from bottom at tunnel exit.
prokaryotes.
small
subunit
Sec61 channel
large
subunit
path of
nascent
protein
The cutaway view at right shows that the tunnel in the
yeast large ribosome subunit, through which nascent
polypeptides emerge from the ribosome, lines up with the
lumen of the ER Sec61 channel.