Transcript Translation

mRNA, tRNA y rRNA
CA García Sepúlveda MD PhD
Laboratorio de Genómica Viral y Humana
Facultad de Medicina, Universidad Autónoma de San Luis
Potosí
1
Contents
• Central Dogma
• mRNA
– Prokaryote
– Eukaryote
– Extrachromosomal (Mitochondrial)
•
•
•
•
•
tRNA
Translation
mRNA Life Cycle
Polycistronic and monocistronic mRNAs
Prokaryotic and eukaryotic mRNAs
2
Central Dogma
• Genetic information is expressed in two stages:
1.- Transcription
2.- Translation
• Generation of ssRNA from
DNA template
• Catalysed by RNA
Polymerases
• Generates:
• mRNA
• tRNA
• rRNA
• Occurs in prokaryotes and
eukaryotes by essentially
identical processes
3
Central Dogma
• Genetic information is expressed in two stages:
1.- Transcription
2.- Translation
• Synthesis of a protein
• mRNA as a template
• tRNAs convert codon
information into amino acid
sequence
• Catalysed by rRNA
4
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
5
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
6
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
7
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
8
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
9
mRNA
• mRNAs are single
stranded RNA
molecules
• They are copied from
the TEMPLATE strand
of the gene, to give
the SENSE strand in
RNA
• They are transcribed
from the 5’ to the 3’
end
• They are translated
from the 5’ to the 3’
end
10
mRNA Prokaryotes
• They can code for one or many proteins (translation of products) in
prokaryotes (polycistronic)
11
mRNA Eukaryotes
• They encode only one
peptide(each) in eukaryotes
(monocistronic).
• Polyproteins are observed in
eukaryotic viruses, but these
are a single translation
product, cleaved into separate
proteins after translation.
12
mRNA Synthesis (transcription)
• Catalysed by RNA Polymerase
• Stages: initiation, elongation and
termination
• Initiation is at the Promoter sequence
• Regulation of gene expression is at
the initiation stage
• Transcription factors binding to the
promoter regulate the rate of
initiation of RNA Polymerase
13
mRNA Synthesis (Prokaryotes)
• 5’ and 3’ ends are unmodified
• Ribosomes bind at ribosome binding
site (do not require a free 5’ end)
• Can contain many open reading
frames (ORFs)
• Translated 5’  3
• Transcribed and translated together
14
mRNA Life Cycle
• mRNA is synthesised by RNA Polymerase
• Translated (once or many times)
• Degraded by RNAses
• Steady state level depends on the rates of both
synthesis and degradation
Production  Degradation
15
mRNA Life Cycle
• mRNA is synthesised by RNA Polymerase
• Translated (once or many times)
• Degraded by RNAses
• Steady state level depends on the rates of both
synthesis and degradation
Production  Degradation
16
mRNA Life Cycle
• mRNA is synthesised by RNA Polymerase
• Translated (once or many times)
• Degraded by RNAses
• Steady state level depends on the rates of both
synthesis and degradation
Production  Degradation
17
mRNA Life Cycle
• mRNA is synthesised by RNA Polymerase
• Translated (once or many times)
• Degraded by RNAses
• Steady state level depends on the rates of both
synthesis and degradation
Production  Degradation
18
mRNA, Eukaryotes
•Linear RNA structure
•5’ and 3’ ends are modified
•5’ GpppG cap
•3’ poly A tail
•Transcribed, spliced,
capped, poly Adenylated in
the nucleus, exported to the
cytoplasm
19
Translation in Eukaryotes
• Translated from 5’  3’ end
in cytoplasm
• Ribosomes bind at 5’ cap,
and do require a free 5’ end
• Can contain only one
translated open reading
frames (ORF).
• Only first open reading
frame is translated
20
Translation in Eukaryotes
• Translated from 5’  3’ end
in cytoplasm
• Ribosomes bind at 5’ cap,
and do require a free 5’ end
• Can contain only one
translated open reading
frames (ORF).
• Only first open reading
frame is translated
21
Translation in Eukaryotes
• Translated from 5’  3’ end
in cytoplasm
• Ribosomes bind at 5’ cap,
and do require a free 5’ end
• Can contain only one
translated open reading
frames (ORF).
• Only first open reading
frame is translated
22
mRNA, 5' Capping
•The 5' cap is a specially altered
nucleotide end to the 5' end of
precursor messenger RNA and, as a
special exception, caliciviruses
(Norwalk virus).
•Actue epidemic gastroenteritis
(nursery, hospital and cruises).
} 30 nm
23
mRNA, 5' Capping
• Consists of a guanine nucleotide
connected to the mRNA via an
unusual 5' to 5' triphosphate
linkage.
• This guanosine is methylated on C7
position directly after capping by a methyl
transferase.
• It is referred to as a 7-methylguanosine cap,abbreviated m7G.
24
mRNA, 5' Capping
• Further modifications include the
methylation of the 2' hydroxy-groups
of the first 3 ribose sugars of the 5'
end of the mRNA.
• Functionally the 5' cap looks
like the 3' end of an RNA
molecule (the 5' carbon of the
cap ribose is bonded, and the 3'
unbonded).
• Offers resistance to 5'
exonucleases.
25
mRNA, 5' Capping process
1) One of the terminal phosphate
groups is removed (phosphatase),
leaving two terminal phosphates.
2) GTP is added to the terminal
phosphates (guanylyl transferase),
losing two phosphate groups (from
the GTP) in the process. This results
in the 5' to 5' triphosphate
linkage.
3) The guanine is methylated (by a
methyl transferase).
4) Other methyltransferases are
optionally used to carry out
methylation of 5' proximal
nucleotides.
26
mRNA, 5' Capping process
1) One of the terminal phosphate
groups is removed (phosphatase),
leaving two terminal phosphates.
2) GTP is added to the terminal
phosphates (guanylyl transferase),
losing two phosphate groups (from
the GTP) in the process. This results
in the 5' to 5' triphosphate
linkage.
3) The guanine is methylated (by a
methyl transferase).
4) Other methyltransferases are
optionally used to carry out
methylation of 5' proximal
nucleotides.
27
mRNA, 5' Capping process
1) One of the terminal phosphate
groups is removed (phosphatase),
leaving two terminal phosphates.
2) GTP is added to the terminal
phosphates (guanylyl transferase),
losing two phosphate groups (from
the GTP) in the process. This results
in the 5' to 5' triphosphate
linkage.
3) The guanine is methylated (by a
methyl transferase).
4) Other methyltransferases are
optionally used to carry out
methylation of 5' proximal
nucleotides.
28
mRNA, 5' Capping process
1) One of the terminal phosphate
groups is removed (phosphatase),
leaving two terminal phosphates.
2) GTP is added to the terminal
phosphates (guanylyl transferase),
losing two phosphate groups (from
the GTP) in the process. This results
in the 5' to 5' triphosphate
linkage.
3) The guanine is methylated (by a
methyl transferase).
4) Other methyltransferases are
optionally used to carry out
methylation of 5' proximal
nucleotides.
29
mRNA, 5' Capping process
1) The Capping Enzyme Complex (CEC) is bound
to the RNA polymerase II before transcription
starts.
2) As soon as the 5' end of the new transcript
emerges the enzymes transfer to it and caps it.
3) The enzymes for capping can only bind to RNA
polymerase II.
30
mRNA, 3' Polyadenylation
Covalent linkage of a poly(Adenine) tail to most
mRNA (eukaryotic).

The poly-A tail protects the mRNA from
exonucleases and is important for transcription
termination, for export of the mRNA from the
nucleus, and for translation.

Some prokaryotic mRNAs also are
polyadenylated.

Polyadenylation occurs after transcription of
DNA into RNA in the nucleus.

Poly-A signal is transcribed, mRNA is cleaved
(endonuclease) in a special site (AAUAAA) then
50 to 250 adenine residues are added.

This reaction is catalyzed by polyadenylate
polymerase.

31
mRNA, 3' Polyadenylation
Cleavage and Polyadenylation Specificity
Factor (CPSF) & Cleavage Stimulation
Factor (CstF), both of which are multiprotein complexes, start bound to the rear
of the advancing RNA polymerase II.
32
mRNA, 3' Polyadenylation
As RNA polymerase II advances over the
adenylation signal sequence, CPSF &
CstF transfer to the pre-mRNA.
CPSF binds to the AAUAAA sequence.
CstF binds to the GU or U rich sequence
following it.
33
mRNA, 3' Polyadenylation
CPSF & CstF promote cleavage 35 nt after
AAUAAA sequence.
Polyadenylate Polymerase (PAP)
immediately starts adding Adenines.
Cleavege does not take place before PAP
has bound.
Nuclear Polyadenylate Binding Protein
(PABPN1) immediately binds to the new
poly-A tag.
34
mRNA, 3' Polyadenylation
PAB displaces CPSF.
PAP continues to add 100 – 250
adenines (depending on the organism).
PABPN1 acts as a molecular ruler, specifying
when polyadenylation should stop.
35
Mitochondrial mRNA = Prokaryote
•
•
•
•
Single stranded
Polycistronic (many ORFs)
Unmodified 5’ and 3’ ends
Transcribed and translated
together
• Show similarity to prokaryote genes
and transcripts
36
tRNA
• tRNA first hypothesized by Francis Crick.
• Small RNA chain that transfers a specific amino
acid to a growing polypeptide chain in the
ribosome
• Has a 3' terminal site for amino acids (whose
linkage depends on aminoacyl tRNA
synthetase).
• Contains a three base region called the
anticodon that complements the codon on the
mRNA.
• Each type of tRNA molecule can be attached to
only one type of amino acid.
• tRNA molecules bearing different anticodons
may also carry the same amino acid
(degenerecy).
37
tRNA Structure
• tRNA has primary structure (sequence), secondary structure (cloverleaf), and
tertiary structure (L-shape).
• Small RNAs 75 - 85 bases in length
• Highly conserved secondary and tertiary structures
• Each class of tRNA charged with a single amino acid
• Each tRNA has a specific trinucleotide anti-codon for mRNA recognition
• Conservation of structure and function in prokaryotes and eukaryotes
38
tRNA Structure
1) The 5'-terminal phosphate group.
2) The acceptor stem (7bp) composed by the 5'-terminus
base paired to the 3'-terminus (contains non-WatsonCrick base pairs).
3) The CCA tail is at the 3' end of the tRNA molecule
(important for the recognition of tRNA by enzymes critical
in translation). NOTE: In prokaryotes, the CCA sequence
is transcribed. In eukaryotes, the CCA sequence is
added.
4) The D arm (18bp) ends in a loop which contains
dihydrouridine.
5) The anticodon arm (ca17bp) contains the anticodon.
6) The T arm (17bp) contains TC sequence
( = pseudouridine).
7) Modified (methylated) bases occur in several positions
outside the anticodon. First anticodon base sometimes
modified to inosine or ).
39
tRNA Structure
40
tRNA Codons & Anti-codons

An anticodon is a unit made up of three nucleotides that complement the
three bases of the codon on the mRNA.
41
tRNA Codons & Anti-codons

Different base triplets (codons) in mRNA code for different amino acids.
42
tRNA Codons & Anti-codons

The Genetic Code is degenerate
43
tRNA Codons & Anti-codons





The Genetic Code is degenerate
Serine can be: UCU, UCC, UCA, UCG, AGU
& AGC
Each tRNA contains a specific anticodon
triplet sequence that can base-pair to one or
more codons for an amino acid.
For example, one codon for Serine is AGU;
the anticodon being UCA.
Some anticodons can pair with more than one
codon due to wobble base pairing. When the
first nucleotide of the anticodon is either
inosine or pseudouridine (can hydrogen bond
to different bases).
44
tRNA Aminoacylation





Aminoacylation is the process of
covalently adding an aminoacyl
group to a compound tRNA).
Each tRNA is aminoacylated
(charged) with a specific amino acid
by an aminoacyl tRNA synthetase.
There is normally a single aminoacyl
tRNA synthetase for each amino
acid.
There can be more than one tRNA,
and more than one anticodon, for an
amino acid.
Recognition of the tRNA by the synthetases depends on the anticodon, and
the acceptor stem.
45
tRNA genes

Organisms have varying amounts of tRNA genes.

C. elegans has 29,647 genes of which 620 code for tRNA.

Saccharomyces cerevisiae has 275 tRNA genes in its genome.

In the human genome there are:

4,421 non-coding RNA genes (which include tRNA genes).

22 mitochondrial tRNA genes

497 nuclear genes encoding cytoplasmic tRNA molecules and

324 tRNA-derived putative pseudogenes.
46
tRNA genes

Cytoplasmic tRNA genes are grouped into 49 families according to their anticodon features.

tRNA genes are found on all chromosomes, except 22 and Y.

High clustering on 6p and 1 is observed (140 tRNA genes).


tRNA molecules are transcribed (in eukaryotic cells) by RNA polymerase III,
unlike messenger RNA which is transcribed by RNA polymerase II.
pre-tRNAs contain introns; in bacteria these self-splice, whereas in eukaryotes
and archaea they are removed by tRNA splicing endonuclease.
47
Ribosomes

Prokaryotic ribosomes are smaller than most of the eukaryotes.

The ribosomes in eukaryote mitochondria resemble those in bacteria.

The function of ribosomes is the assembly of proteins (translation).

Ribosomes catalyze the assembly of individual amino acids into polypeptide
chains.

They use mRNA as a template to join a correct sequence of amino acids.

This reaction uses adapters called tRNA.
48
Ribosomes




First observed in the mid-1950s by Romanian cell
biologist George Palade using an electron microscope as
dense particles or granules
Nobel Physiology, 1974.
The term "ribosome" was proposed
by scientist Richard B. Roberts in
1958
Ribonucleic body (soma)
49
Ribosomes

All ribosomes are composed of two subunits that separate when
translation terminates and reunite when an new initiation complex is
formed.
Small subunit
mRNA
tRNA
Large subunit
50
Session #20-22 Translation
Introduction
• The basic form of the ribosome is conserved, but there are appreciable variations
in the overall size and proportions of RNA and protein in the ribosomes of
bacteria, eukaryotic cytoplasm, and organelles.
Al ribosomes
consists of two
subunits, each of
which contains a
major rRNA and a
number of small
proteins.
The large subunit
may also contain
smaller RNAs.
51
Svedberg Units Briefly







Theodor Svedberg (1884-1971).
Nobel laureate (Chemistry,1926) for his work on
colloids and ultracentrifugation.
Colloid = Mechanical mix (milk)
with a dispersed and continuous
phase.
Svedberg, non-SI unit used to characterize the behaviour of a
particle in ultracentrifugation.
S = unit of time amounting to 10-13 s or 100 femtoseconds.
S not additive, since the sedimentation rate is associated with the shape & size of
the particle.
When two particles bind together there is a loss of surface area, when measured
separately they will have Svedberg values that do not add up.
52
Session #20-22 Translation
Introduction
• The ribosomal proteins are known as r-proteins.
• With the exception of one protein present at four copies per ribosome, there is
one copy of each protein.
53
Session #20-22 Translation
Phylogenetics
• The ribosomes of higher
eukaryotes are larger than those
of bacteria.
• Total content of both RNA &
protein is greater
• Major RNA molecules are longer
(18S & 28S rRNAs)
• Possess more proteins.
• RNA is the predominant
component.
54
Session #20-22 Translation
Phylogenetics
• The ribosomes of higher
eukaryotic cytoplasm are larger
than those of bacteria.
• Total content of both RNA &
protein is greater
• Major RNA molecules are longer
(18S & 28S rRNAs)
• Possess more proteins.
• RNA is the predominant
component.
55
Session #20-22 Translation
Phylogenetics
• The ribosomes of higher
eukaryotic cytoplasm are larger
than those of bacteria.
• Total content of both RNA &
protein is greater
• Major RNA molecules are longer
(18S & 28S rRNAs)
• Possess more proteins.
• RNA is the predominant
component.
56
Session #20-22 Translation
Phylogenetics
• In prokaryotes:
– Ribosomes = 2.5 MDa
– rRNA = 66% of the ribosomal mass
• In eukaryotes (mammals):
– Ribosomes = 4.2 MDa
– rRNA = 60% of the ribosomal mass
• Organelle ribosomes are distinct from
eukaryotic cytosol ribosomes and take
varied forms.
• In some cases, they are almost the size of bacterial ribosomes and have
70% RNA; in other cases, they are only 60S and have <30% RNA.
57
Ribosomes

Prokaryotic Ribosome is 70S and composed of Large (50S) and Small (30S)
subunits.


Large subunit composed of:

23S rRNA (2900 nt)

5S rRNA (120 nt)

31-34 proteins.
Small subunit composed of:

16S rRNA (1540 nt)

21 proteins.
58
Ribosomes


Eukaryotic Ribosome is 80S and composed of Large (60S) and Small
(40S) subunits.
Large subunit composed of:





5.8S rRNA (160 nt)
28S rRNA (4700 nt)
5S rRNA (120 nt)
~49 proteins
Small subunit composed of:


18S rRNA (1900 nt)
~33 proteins
59
Ribosomes

The ribosomes of chloroplasts & mitochondria also consist of large and
small subunits bound together with proteins into one 70S particle (as
prokaryotes do).
60
Ribosomes

Ribosomes are about 20nm in diameter.

Can be studied with EM.
Curiously, rRNA genes in a cluster !
61
Session #20-22 Translation
Transmission Electron Microscopy
• The complete 70S ribosome has an asymmetric
construction.
• The partition between the head and body of the
small subunit is aligned with the notch of the
large subunit, so that the platform of the small
subunit fits into the large subunit.
• There is a cavity between the subunits which
contains important sites.
62
Ribosome location
Classified as "free" or "membrane-bound".

Free ribosomes move about the cytoplasm
(within the cell membrane).


Proteins formed from free ribosomes
are used within the cell.
Membrane-bound ribosomes place newly
produced polypeptides directly into the
endoplasmic reticulum.


Usually produce proteins that are used within the cell membrane or are
expelled from the cell via exocytosis.
Proteins containing disulfide bonds using cysteine cannot be produced
outside of the lumen of the endoplasmic reticulum.
63
Ribosomes

Composed of 60% ribosomal RNA and 30% ribosomal proteins (known
as a Ribonucleoprotein or RNP).
PyMol
64
Ribosomes


Crystallographic work has shown
that there are no ribosomal
proteins close to the reaction site
for polypeptide synthesis.
This suggests that the protein
components of ribosomes act as
a scaffold that may enhance the
ability of rRNA to synthesize
protein rather than directly
participating in catalysis.
65
Ribosomes

Their active sites (A, P & E) are made of RNA, so ribosomes are now
classified as "ribozymes".
66
tRNA binding to ribosome

The ribosome has three binding sites for tRNA
molecules: the A, P and E sites.

A= aminoacyl-tRNA site

P= Peptidyl-tRNA site

E= Egress site
67
tRNA binding to ribosome

The ribosome has three binding sites for tRNA
molecules: the A, P and E sites.




A= aminoacyl-tRNA site

P= Peptidyl-tRNA site

E= Egress site
During translation the A site binds an incoming
aminoacyl-tRNA as directed by the codon
currently occupying this site.
This codon specifies the next amino acid to be
added to the growing peptide chain.
The A site is only working after the first
aminoacyl-tRNA has attached to the P site.
68
tRNA binding to ribosome

The ribosome has three binding sites for tRNA
molecules: the A, P and E sites.




A= aminoacyl-tRNA site

P= Peptidyl-tRNA site

E= Egress site
The P-site codon is occupied by peptdyl-tRNA (a
tRNA with multiple amino acids attached).
The P site is actually the first to bind to
aminoacyl tRNA.
This tRNA in the P site carries the chain of
amino acids that has already been synthesized.
69
tRNA binding to ribosome

The ribosome has three binding sites for tRNA
molecules: the A, P and E sites.


A= aminoacyl-tRNA site

P= Peptidyl-tRNA site

E= Egress site
The E site is occupied by the empty tRNA as it is
about to exit the ribosome.
70
tRNA binding to ribosome

The ribosome has three binding sites for tRNA
molecules: the A, P and E sites.


A= aminoacyl-tRNA site

P= Peptidyl-tRNA site

E= Egress site
The E site is occupied by the empty tRNA as it is
about to exit the ribosome.
Agrawal animation
Sannuga animation
71
Ribosomes

Their active sites (A, P & E) are made of RNA, so ribosomes are now classified as
"ribozymes".
PyMol
72
Ribosomes




Prokaryote and eukaryote ribsome differences lie outside of functional
parts (A, P & E sites) and therefore “redundant insertions”.
The differences are exploited by pharmaceuticals to create antibiotics that
destroy a bacterial without harming the cells of the infected person.
Even though mitochondria possess similar ribosomes they are not
affected by these antibiotics (double membrane).
Antibiotics such as macrolides, aminoglycosides and others:







anisomycin
cycloheximide
chloramphenicol
tetracycline
streptomycin
erythromycin
puromycin, etc.
73
Ribosomes & Antibiotics




Structure of the antibiotic gentamicin
C1a bound to its rRNA target.
Gentamicin, an aminoglycoside
antibiotic, binds within the major
groove of the RNA, which is located
in the decoding site of the bacterial
ribosome.
Aminoglycosides cause misreading
of the genetic code.
Binding of the drug causes a
conformational change in ribosomal
RNA that disrupts high-fidelity
reading of the genetic code.
74
Ribosomes & Antibiotics



Structure of the aminoglycoside
paromomycin bound to the bacterial
rRNA decoding site.
The sites that lead to resistance are
highlighted with purple spheres.
The N7 methylation at G1405 only
causes resistance to aminoglycosides
like gentamicin that contact this
position directly.
75
Ribosomes& Translation

Ribosomes are the workhorse for protein biosythesis= TRANSLATION
76