P1253814587lqsGU - PowerPoint PPT Presentation

Title: P1253814587lqsGU


1
XII. Transcription and Translation   Expression
is an information flow from DNA to
Protein.   The question is that how the
information stored in DNA is decoded into a
protein.  
2
Basic Structure of Protein   Proteins are formed
by linear polymers of amino acids.  
Peptide bonds link up individual amino acids to
form a polypeptide chain, which grows from the
N terminal end to the C terminal end.  
3
(No Transcript)
4
The basic principle is that one protein product
is coded by one gene. A gene can also be
called a cistron or an open reading frame.  
5
How is the genetic information encoded?
(338-342)   The genetic code is written in units
of three letters of nucleotides called codon.
Each triplet in DNA (RNA) sequence is a word
that codes for a specific amino acid in the
protein product.  
6
Exp. DNA 5- CTG CGC TAC TCT AAA 3
(transcription)   mRNA 5- CUG CGC
UAC UCU AAA- 3 (translation)
  Protein Leu Arg Tyr Ser
Lys  
DNA expression has two steps.
7
After transcription and translation, the genetic
information stored in DNA is decoded into amino
acid sequence which is a polypeptide chain or a
protein.   It is the different combination of
four different nucleotides that specifies 20
different amino acids in a protein.  
8
The genetic code exhibits a number of
characteristics   1. The genetic code is
written in linear form, each triplet in
mRNA is a codon that specifies one amino
acid.   2. The code contains "start" and "stop"
signals, which are responsible for the
initiation and termination of translation.
For exp., AUG - start codon, UAG - stop
codon.  
9
3. The code is degenerate. More than one codons
can be assigned to a single amino
acid.    Exp. DNA 5- CTG CGC TAC TCT AAA
3 TCC
(transcription)   mRNA 5- CUG CGC UAC
UCU AAA- 3 UCC
(translation)   Protein Leu Arg
Tyr Ser Lys
There are 20 amino acids. Different combination
of four nucleotides give rise to 64 codons
10
4. The code is nearly universal. The same coding
dictionary is used by all different
species.  
The codon dictionary   The coding dictionary
contains 64 triplets functioning in protein
translation. Among the 64, 61code for 20 amino
acids, 3 are termination signal.
11
Degeneracy and Wobble Hypothesis   One amino
acid can have more than one codons. Among the
total 20 amino acids, there are18 of them can be
coded by one codons. Some (Ser) are even coded
by six codons. In many cases, among these
interchangeable codons, the first two bases are
the same, only the last one is degenerate.
Frances Crick first predicted this in his Wobble
Hypothesis in 1966.  
12
(No Transcript)
13
The biological significance The degenerate
coding system minimizes chance of deleterious
DNA mutation. Changing of one or two
nucleotides by mutation may not always cause
phenotype change.
14
Amino acids with similar chemical characteristics
are often represented by related codons. This
also helps to minimize the effect of mutation.
For exp., Met (AUG) and isoleucine (AUC) both
have long side chain Arg (AGG) and Lys (AAG)
are both basic
15
If the first nucleotide within a codon is
replaced, the result is usually dramatic. For
exp., human growth hormone is a protein with 192
amino acids and is coded by a gene with 600
bp. Glycine residue at 120 position is coded by
codon GGG. A point mutation that switch GGG
into AGG would change the amino acid from Gly to
Arg. The biological function of the molecule is
totally changed.
16
Test the biological effects of the mutation by
making transgenic animals (animals that
recombinant DNA).
The mutant DNA and the wild type GH gene was
purified and microinjected into the fertilized
mouse egg to generate transgenic mice
17
Micro Injection to Produce Transgenic Mice
18
(No Transcript)
19
The degenerate coding system requires more than
20 different tRNA species for translation.
  Current estimate 30-40 tRNA In bacteria,
about 50 in animals.
20
Codon usage   Although many of the amino acids
have more than one codon assignments, each of
the alternative codons are not used in equal
frequency. Especially in different species,
the codon usage for same amino acids can be
apparently different.  
21
(No Transcript)
22
The practical significance of codon usage
application of gene transfer between organisms
and species may be limited.
23
Initiation, termination and suppression   The
initiation codon in mRNA is AUG which codes for
methionine.   In bacteria, the initial amino
acid is always a formylated methionine
(N-formylmethionine). In eukaryotic cells, this
methionine is not formylated.   There are three
triplets serve as termination codons (UAG, UAA,
and UGA). They cannot be recognized by tRNA.
24
Nonsense mutation - A mutation that can cause
the internal codons change into a stop codon,
which can cause the premature termination of
translation.   Suppression - a secondary
mutation that suppresses the premature
termination. These mutations cause the
termination signal to be read as a sense
codon. A correction mechanism then fill in an
amino acid that is usually different from the
one in the wild type.  
25
The end protein product may be not altered
drastically and may function almost normally.
26
Transcription -The passing of information from
DNA to RNA.   Transcription is the synthesis of
RNA using DNA as template. The enzyme that does
the RNA synthesis is called RNA polymerase.
  Three major types of RNA molecules mRNA
which determines the amino acid sequence tRNA
that transfers amino acids during protein
synthesis, and rRNA that is associated with
ribosome.  
27
Transcription in prokaryotes   Transcription
requires a template DNA, RNA polymerase, and
NTPs (ATP, UTP, GTP, CTP).  
28
Bacterial RNA polymerase is a polymer containing
six subunits, two ?, one ?, one ?, one ?, and
one ?. ?, ?, ?, and ? are tightly bound to
each other and form a core enzyme that can do
RNA synthesis. The ? subunit is required to
recognize the starting point of transcription.
It can bind to DNA and the core enzyme to form a
holoenzyme.
29
The DNA sequence that ? subunit recognizes is
called promoter. The DNA sequence in promoter
region remains similar between different genes
and is, therefore, called consensus sequence.
It includes two short stretches (TA) located at
-35 and 10. (35 or 10 bp upstream to the
starting point).     1 is the transcription
starting site. There is no 0. (Fig. 11.8)
30
(No Transcript)
31
(No Transcript)
32
Transcription Termination Signals the DNA
sequence that tell RNA polymerases to stop
working. Two types of transcription
termination 1. ? Independent The termination
signal consists of two symmetrical 8bp GC
sequences followed by a stretch of AT rich
sequence.
33
The two symmetrical GC rich sequences can form a
hairpin loop to allow the RNA chain to dissociate
from DNA template. (Fig. 11.10) The AT rich
sequence also helps the dissociation Between
mRNA and the DNA template.
34
2. ?-Dependent Termination Some times
transcription termination requires a protein
called ? factor to dissociate RNA and
RNA polymerase from the DNA template. The
termination signal sequence does not include the
AT rich run.
35
The DNA that serves as the template for
transcription is the one that reads from 3' -
5. The other strand that runs 5 3 is
called coding strand (sequence). The
orientation of RNA transcript is same as the
coding strand. The sequences between the
transcription beginning and transcription
termination is called a gene, which contains the
information to make a polypeptide.
36
The other name for a gene is sistron. In
bacteria and viruses. One mRNA can contain
multiple sistrons polysistron. More than one
polypeptide can be coded by a single message.  
37
(No Transcript)
38
Transcription in eukaryotes    1.
Transcription in eukaryotes occurs in nucleus,
(mRNA must move into the cytoplasm to be
translated)   2.  Eukaryotes have three
different RNA polymerases   3.  Eukaryotic mRNA
do not have multiple sisitrons,
but...   4.  The maturation of eukaryotic mRNA
involves post-transcriptional processing.
 
39
Three RNA polymerases in eukaryotes   RNA Pol. I
- responsible for the synthesis of ribosomal
RNA RNA Pol. III - responsible for tRNA and 5s
rRNA, and some of the snRNA RNA Pol. II -
responsible for mRNA and some snRNA    
40
RNA Pol.II is responsible for the synthesis of
the precursor of mRNA or pre-mRNA. Pre-mRNA
is also called heterogeneous nuclear RNA, which
has to be excised to become mRNA.
41
The eukaryotic promoter sequence is called TATA
box which is a consensus sequence of TATAAA
located at 25. The other two consensus
sequences that also affect gene transcription
are CAAT box and GC box located near 80
position.  
42
(No Transcript)
43
Post-Transcriptional Modification   There are
three major alterations happen to the precursor
mRNA. 1). The 5 end capping 2). The 3 end
poly A addition. 3). Slicing The events occur
in the nucleus in the above order.
4). RNA editing
44
1). 5 end capping The addition of a
7-methylguanosine (7mG) cap to the 5 end of the
transcript.  
45
(No Transcript)
46

• Possible role of the 5 end cap
• protect the 5 end from nuclease attack
• help the transport of the transcript from
  nucleus
• to the cytoplasm.

47

• 2). 3 end polyadenylation
• 20 - 250 adenines are added to the 3 end of the
  
• transcript by poly A polymerase.

• This fragment is called poly A tail, and the
  process
• is called polyadenylation.
• Polyadenylation signal AAUAAA. The poly A
• tail is added 10 or 15 bases after this signal
  by poly
• A polymerase.
• The poly A tail is believed to increase the
  stability
• of mRNA.
•  

48
Practical significance of poly A tail To
isolate mRNA (for establishing cDNA
library). Oligo dT (stretches of poly T) are
linked to a solid matrix (sepharose), which is
packed in a column. Total RNA extract from cells
is loaded to the column. Only mRNA that has
poly A tails will be retained in the
column (Fig. 11.14)
49
3). Splicing 1977 Chicken albumin molecule
386 a.a. Chicken albumin gene 7700 bp 386 x 3
1158 ?
There are noncoding sequences of 6542 bp.
50
In eukaryotic genes there are noncoding
sequences called introns, which separate the
coding sequences, exons.
After transcription, a precursor RNA contains
both exons and codons. The process of intron
removal is called splicing. Spliceosome -
RNA-protein complex that participates
splicing
51
Splicing donor site
Splicing receiver site
Intron 1
Spliced out
Introns are usually much larger than exons.
52
(No Transcript)
53
Function of Introns? Possible functions 1). To
absorb mutations 2). May contain sequences that
bound by transcriptional factor. 3).
Ribozyme RNA enzyme that catalyzes splicing
reaction
54
Autocatalytic splicing (p329) Mid 1970s, Sidney
Altman and Thomas Cech discovered that not all
enzymes are proteins. Introns can be spliced out
without any protein elements. RNA fragments
that has enzyme function and catalyze splicing
are called ribozymes.
55
4). RNA editing   The nucleotide sequence of
pre-mRNA can be changed prior to translation.
There can be substitution editing or insertion/
deletion editing.  
56
Exp., mRNA of human apolipoprotein B has two
forms long and short. (One gene encodes two
proteins). In human intestinal cells, ApoB
mRNA is edited by a single C-U substitution.
A CAA, glutamine codon is changed into UAA,
the stop codon. So, the protein synthesis is
terminated at about half sized of the original
form.  
57
RNA editing one of the mechanisms for tissue
specific regulation of gene expression.
58
Translation and Proteins   Translation - mRNA
sequence is converted into amino acid
sequence.   In eukaryotic cells, transcription
and translation are two separated
processes.   Prokaryotes have no nuclei.
Transcription and translation are coupled,
occurring almost simultaneously.  
59
1. Ribosome and tRNA    Ribosome is a complex of
ribosomal proteins and rRNA. Two subunits
Prokaryotic ribosome 50S 30S 70S Eukaryotic
ribosome 60S 40S 80S. S represents
Svedburg units. It is proportional to the
molecules mass. However, there is no linear
relationship between the mass and S value.
60
Each ribosome subunit contains one or more single
stranded RNA complexed with of proteins. The
single stranded RNA chains forms multiple loops
and hairpin structure. The self folding and
base pairing can increase the stability of the
molecule. mRNA does not have such a secondary
structure. This may be why its not as stable
as rRNA or tRNA.  
61
(No Transcript)
62
In association with a ribosome, mRNA presents a
triplet codon that calls for a specific amino
acid. The mechanism that links up mRNA and
amino acids is the tRNA which works as an
adapter to recognized the appropriate codon on
mRNA.
63
tRNA is a small molecule with 75-90
nucleotides that forms a cloverleaf
structure. An important feature of tRNA is its
anticodon structure which contains three bases
that are complementary to the codon on mRNA and
can base pair with the codon. The opposite
region (3 end) of the molecule is the binding
site for amino acids.  
64
(No Transcript)
65
(No Transcript)
66
Theoretically, there should be 61 different
types of tRNA each has anticodon complementary
to the correspondent codon on mRNA. For Exp.,
alanine has 3 codons, GCU, GCC and GCA. But
the anticodon found for these triplets turned
out to be one, CGI. I inosine, a modified
nucleotide that can form H-bond with U, C and A.

67
So, I is a wobble position in tRNA molecule.
The existence of the wobble structure in tRNA is
perhaps to increase the translation efficiency.
The actual number of tRNA is less than 61.
How many exactly? Current estimate 30 -40 in
bacteria and up to 50 in animals and plants.  
68
Protein synthesis   The first step is the
attachment of amino acids to their specific
tRNAs amino acid activation  
This attachment is carried out by an enzyme
family with at least 20 members called aminoacyl
tRNA synthetases. For example, the enzyme for
proline is called prolyl-tRNA synthetase.
This reaction requires ATP as energy source and
Mg as cofactor. )
69
After attachment, the tRNA will bring the proper
amino acid to the specific codon on mRNA.
70
So, I is a wobble position in tRNA molecule.
The existence of the wobble structure in tRNA is
perhaps to increase the translation efficiency.
The actual number of tRNA is less than 61.
How many exactly? Current estimate 30 -40 in
bacteria and up to 50 in animals and plants.  
71
Protein synthesis   The first step is the
attachment of amino acids to their specific
tRNAs amino acid activation  
This attachment is carried out by an enzyme
family with at least 20 members called aminoacyl
tRNA synthetases. For example, the enzyme for
proline is called prolyl-tRNA synthetase.
This reaction requires ATP as energy source and
Mg as cofactor. )
72
After attachment, the tRNA will bring the proper
amino acid to the specific codon on mRNA.
73
  The initiation step - recognition of the
initiation codon. The first tRNA with an
attached amino acid binds to the P site (or
peptidyl site) on the ribosome. The first
codon is almost always an AUG that codes for a
methionine. So it is recognized by met-tRNAinit
.  
74
(No Transcript)
75

• Chain elongation - always from N terminus to C
• terminus.
• the entering of an aminoacyl-tRNA into A site,
• then, a peptide bond is formed between the 1st
• a.a. (Met on P site) and the 2nd a.a. attached
• to the A site.
• This reaction is catalyzed by peptidyl
  transferase.
• In the mean time the covalent bond between 1st
  a.a.
• and tRNAinit is hydrolyzed.

76
(No Transcript)
77
Then, tRNAinit is released from the P site and
tRNA- dipeptide complex transfer from A site to P
site. In the mean time the ribosome is moving
down stream on mRNA or the mRNA advances through
the ribosome.
78
Termination   Three termination codons, UAG, UAA,
and UGA do not specify any amino acids and
therefore have no complementary tRNAs. When
protein synthesis comes to one of these codons,
translation is terminated. In E.coli, the
enzymes that control translation termination are
known as release factors RF1 and RF2. In
eukaryotes, there is only one named eRF.  
79
Open reading frame and 5 UTR The DNA or mRNA
sequences between start codon and stop codon is
called open reading frame. Note, the
initiation codon is not the first codon on mRNA.
There are 20 1000 bases between the cap site
and the translation initiation site, depending
upon the gene. The region in mRNA before the
start codon is called leading region or 5
untranslated region (5 UTR).  
80
In bacteria and viruses, some genes may code for
more than one protein products. There may be
more than one start or stop codons. When the
sequence between initiation and termination
codons remains three by three arrangement, we
say its in frame and the information is
called an open reading frame.  
81
Overlapping genes   It is possible for a single
gene to have multiple start points stop codons
for translation. If so, a same mRNA can have
more than one open reading frames that care
called overlapping genes. OLG is very often
seen in viruses and bacteria. The use of
overlapping genes allow viruses to produce
enough protein products on the bases of limited
amount nucleic acids high efficiency.  
82
(No Transcript)
83
Translation is more complex in eukaryotes.   1.
Transcription and translation are not coupled.
For translation to occur, mRNA has to migrate
out of nucleus into the cytoplasm to be
associated with ribosome.   2. Most ribosomes
are associated with endoplasmic reticulum, which
is the place that protein trafficking and post
translational modification occur. The newly
synthesized polypeptide is secreted by ribosome
into the ER system.  
84
3.  Signal peptide Most eukaryotic proteins
have about 30 a.a. signal peptide sequence at
the N terminus. It determines the destination
of the protein inside the cell. For exp., for
a protein to be secreted out of the cell, its
signal peptide will lead it into ER, a protein
trafficking system. This signal peptide will be
cleaved while the protein enters the ER.  
85
(No Transcript)