DNA Transcription and Translation

1.

DNA Transcription and
Translation

2.

Comparing RNA and DNA
DNA can replicate itself precisely and contain
information in the specific sequence of its
bases.
RNA and DNA are very similar molecules.
5-carbon sugar in RNA is Ribose
RNA contains Uracil instead of Thymine
DNA is double stranded and RNA is single
stranded with folded complex secondary and
tertiary structures.
DNA molecules are always longer than RNA
molecules.
DNA is more stable than RNA.
There are several classes of RNA.

3.

Messenger RNA
mRNA carries the message. The linear amino acid
sequence (primary) is encoded in the DNA. But the
DNA does not make the proteins directly. mRNA is
the link between gene and protein.
The information contained in the mRNA is written in
the genetic code. The genetic code is UNIVERSAL.
The beginning of the mRNA is always on the 5’ end
and that is where the synthesis of the proteins starts.
It takes three nucleotides to code for one amino acid.
The mRNA has a 5’ leader, a coding region, introns,
and a 3’ trailer.
After transcription the mRNA is modified to go inside
the cytoplasm. A 5’ cap is added as well as a 3’ poly
A tail. Introns are spliced out. Only expressed regions
(exons) are kept.

4.

Basic Principles of Transcription and
Translation
RNA is the intermediate between genes and the
proteins for which they code
Transcription is the synthesis of RNA under the
direction of DNA
Transcription produces messenger RNA (mRNA)
Translation is the synthesis of a polypeptide, which
occurs under the direction of mRNA
Ribosomes are the sites of translation
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.

EXPERIMENT
No growth:
Mutant cells
cannot grow
and divide
Growth:
Wild-type
cells growing
and dividing
Minimal medium
RESULTS
Classes of Neurospora crassa
Wild type
Class I mutants
Class II mutants
Class III mutants
Minimal
medium
(MM)
(control)
Condition
Fig. 17-2
MM +
ornithine
MM +
citrulline
MM +
arginine
(control)
CONCLUSION
Gene A
Gene B
Gene C
Wild type
Class I mutants
(mutation in
gene A)
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Precursor
Precursor
Precursor
Precursor
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Ornithine
Ornithine
Ornithine
Ornithine
Enzyme B
Enzyme B
Enzyme B
Enzyme B
Citrulline
Citrulline
Citrulline
Citrulline
Enzyme C
Enzyme C
Enzyme C
Enzyme C
Arginine
Arginine
Arginine
Arginine

6.

In prokaryotes, mRNA produced by
transcription is immediately translated
without more processing
• In a eukaryotic cell, the nuclear envelope
separates transcription from translation
• Eukaryotic RNA transcripts are modified
through RNA processing to yield finished
mRNA
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

7.

DNA
TRANSCRIPTION
mRNA
Ribosome
TRANSLATION
Polypeptide
(a) Bacterial cell
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell

8.

The Genetic Code
The genetic code is the same for all
organisms (universal)
A codon is a “word” in DNA/RNA language. It
is formed by three nucleotides.
There are a lot of synonymous codons.
The genetic code is “redundant”
AUG is always the start codon for translation
and there are three stop codons to end
translation.

9.

(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a
jellyfish gene

10.

Key Words and definitions
Transcription describes the synthesis of RNA on a
DNA template
Translation is the synthesis of protein on a mRNA
template
Coding region of part of the gene that represents a
protein sequence.
Codon is a triplet of bases that represents an amino
acid or a termination signal
The antisense strand (template) is complementary
to the sense strand and is used as a template to
synthesize RNA
The coding strand (sense) has the same sequence
as the mRNA and is related to the protein
synthesized.

11.

RNA Polymerase Binding and Initiation of
Transcription
Promoters signal the initiation of RNA
synthesis
Transcription factors mediate the binding
of RNA polymerase and the initiation of
transcription
The completed assembly of transcription
factors and RNA polymerase II bound to a
promoter is called a transcription
initiation complex
A promoter called a TATA box is crucial in
forming the initiation complex in
eukaryotes
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

12.

Promoters, terminators and start
point

13.

1 A eukaryotic promoter
Promoter
5
3
TATA box
Transcription
factors
includes a TATA box
Template
3
5
Template
DNA strand
Start point
2 Several transcription factors must
bind to the DNA before RNA
polymerase II can do so.
5
3
3
5
3 Additional transcription factors bind to
the DNA along with RNA polymerase II,
forming the transcription initiation complex.
RNA polymerase II
5
3
Transcription factors
3
5
5
RNA transcript
Transcription initiation complex

14.

Only one strand of DNA is
transcribed

15.

Sense and antisense strands

16.

17.

Promoter
Transcription unit
5
3
Start point
RNA polymerase
5
3
RNA
transcript
Unwound
DNA
3
5
DNA
1
Initiation
3
5
Template strand
of DNA
2
3 end
3
5
5
5
3 Termination
3
5
5
3
5
RNA nucleotides
5
3
RNA
transcript
RNA
polymerase
3
Elongation
Rewound
DNA
5
3
Nontemplate
strand of DNA
Elongation
Completed RNA transcript
3
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA

18.

G
5
P
P
Protein-coding segment
Polyadenylation signal
P
5 Cap 5 UTR Start codon
AAUAAA
Stop codon
3 UTR
3
AAA … AAA
Poly-A tail

19.

20.

Split Genes and RNA Splicing
Most eukaryotic genes and their RNA transcripts
have long noncoding stretches of nucleotides that lie
between coding regions
These noncoding regions are called intervening
sequences, or introns
The other regions are called exons because they
are eventually expressed, usually translated into
amino acid sequences
RNA splicing removes introns and joins exons,
creating an mRNA molecule with a continuous
coding sequence
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

21.

Pre-mRNA
5 Exon Intron
5 Cap
1
30
Exon
31
Coding
segment
mRNA
5 Cap
1
5 UTR
Exon
Intron
104
105
3
146
Introns cut out and
exons spliced together
Poly-A tail
146
3 UTR
Poly-A tail

22.

In some cases, RNA splicing is carried out
by spliceosomes
Spliceosomes consist of a variety of
proteins and several small nuclear
ribonucleoproteins (snRNPs) that
recognize the splice sites

23.

5
RNA transcript (pre-mRNA)
Exon 1
Protein
snRNA
Intron
Exon 2
Other
proteins
snRNPs

24.

5
RNA transcript (pre-mRNA)
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5

25.

5
RNA transcript (pre-mRNA)
Exon 1
Intron
Exon 2
Protein
snRNA
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
Cut-out
intron
mRNA
Exon 1
Exon 2

26.

Ribozymes
Ribozymes are catalytic RNA molecules
that function as enzymes and can splice
RNA
The discovery of ribozymes rendered
obsolete the belief that all biological
catalysts were proteins

27.

Three properties of RNA enable it to
function as an enzyme
It can form a three-dimensional structure
because of its ability to base pair with itself
Some bases in RNA contain functional
groups
RNA may hydrogen-bond with other
nucleic acid molecules
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

28.

The Functional and Evolutionary Importance of
Introns
Some genes can encode more than one kind of
polypeptide, depending on which segments are
treated as exons during RNA splicing
Such variations are called alternative RNA splicing
Because of alternative splicing, the number of different
proteins an organism can produce is much greater
than its number of genes
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

29.

Proteins often have a modular
architecture consisting of discrete
regions called domains
In many cases, different exons code for
the different domains in a protein
Exon shuffling may result in the
evolution of new proteins
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

30.

Fig. 17-12
Gene
DNA
Exon 1
Intron
Exon 2
Intron
Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide

31.

tRNA

32.

33.

Aminoacyl-tRNA
The aminoacyl-tRNA synthase adds the
correct amino acid to the corresponding
tRNA.

34.

Fig. 17-15-4
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
P
P Pi
Pi
Adenosine
tRNA
Aminoacyl-tRNA
synthetase
Pi
tRNA
P
Adenosine
AMP
Computer model
Aminoacyl-tRNA
(“charged tRNA”)

35.

36.

37.

Ribosomal RNA
Ribosomal RNA:
contributes to the structure
of Ribosomes. In
eukaryotes rRNA is
transcribed exclusively in
the nucleolus. The primary
transcript is processed by
an enzyme to produce the
shorter rRNA constituting
the ribosome.
Ribosomes are made of
40S subunit (small) and
60S subunit (large).
Proteins make up about
half of the ribosome. The
complete ribosome is 80S.

38.

Ribosomes

39.

40.

In Prokaryotes

41.

Fig. 17-16a
tRNA
molecules
Growing
polypeptide
Exit tunnel
Large
subunit
E PA
Small
subunit
5
mRNA
3
(a) Computer model of functioning ribosome

42.

Fig. 17-16b
P site (Peptidyl-tRNA
binding site)
E site
(Exit site)
A site (AminoacyltRNA binding site)
E P A
mRNA
binding site
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
Growing polypeptide
Amino end
Next amino acid
to be added to
polypeptide chain
mRNA
5
E
tRNA
3
Codons
(c) Schematic model with mRNA and tRNA

43.

A ribosome has three binding sites for
tRNA:
The P site holds the tRNA that carries the
growing polypeptide chain
The A site holds the tRNA that carries the next
amino acid to be added to the chain
The E site is the exit site, where discharged
tRNAs leave the ribosome
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

44.

Ribosome Association and Initiation of
Translation
The initiation stage of translation brings
together mRNA, a tRNA with the first amino
acid, and the two ribosomal subunits
First, a small ribosomal subunit binds with
mRNA and a special initiator tRNA
Then the small subunit moves along the
mRNA until it reaches the start codon (AUG)
Proteins called initiation factors bring in the
large subunit that completes the translation
initiation complex
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

45.

Fig. 17-17
3 U A C 5
Met 5 A U G 3
Initiator
tRNA
mRNA
5
P site
GTP GDP
Start codon
mRNA binding site
3
Small
ribosomal
subunit
Met
E
5
Large
ribosomal
subunit
A
3
Translation initiation complex

46.

Elongation of the Polypeptide Chain
During the elongation stage, amino acids are
added one by one to the preceding amino
acid
Each addition involves proteins called
elongation factors and occurs in three steps:
codon recognition, peptide bond formation,
and translocation
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

47.

Amino end
of polypeptide
E
3
mRNA
5
P A
site site

48.

Amino end
of polypeptide
E
3
mRNA
5
P A
site site
GTP
GDP
E
P A

49.

Fig. 17-18-3
Amino end
of polypeptide
E
3
mRNA
5
P A
site site
GTP
GDP
E
P A
E
P A

50.

Fig. 17-18-4
Amino end
of polypeptide
E
3
mRNA
Ribosome ready for
next aminoacyl tRNA
5
P A
site site
GTP
GDP
E
E
P A
P A
GDP
GTP
E
P A

51.

Termination of Translation
Termination occurs when a stop codon in
the mRNA reaches the A site of the
ribosome
The A site accepts a protein called a
release factor
The release factor causes the addition of a
water molecule instead of an amino acid
This reaction releases the polypeptide, and
the translation assembly then comes apart
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

52.

Fig. 17-19-1
Release
factor
3
5
Stop codon
(UAG, UAA, or UGA)

53.

Fig. 17-19-2
Release
factor
Free
polypeptide
3
5
5
Stop codon
(UAG, UAA, or UGA)
3
2 GTP
2 GDP

54.

Fig. 17-19-3
Release
factor
Free
polypeptide
5
3
5
5
Stop codon
(UAG, UAA, or UGA)
3
2 GTP
2 GDP
3

55.

Completing and Targeting the
Functional Protein
Often translation is not sufficient to make a
functional protein
Polypeptide chains are modified after
translation
Completed proteins are targeted to specific
sites in the cell
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

56.

Protein Folding and Post-Translational
Modifications
During and after synthesis, a polypeptide
chain spontaneously coils and folds into its
three-dimensional shape
Proteins may also require post-translational
modifications before doing their job
Some polypeptides are activated by
enzymes that cleave them
Other polypeptides come together to form
the subunits of a protein
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

57.

Targeting Polypeptides to Specific
Locations
Two populations of ribosomes are evident in
cells: free ribsomes (in the cytosol) and
bound ribosomes (attached to the ER)
Free ribosomes mostly synthesize proteins
that function in the cytosol
Bound ribosomes make proteins of the
endomembrane system and proteins that are
secreted from the cell
Ribosomes are identical and can switch from
free to bound
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

58.

Polypeptide synthesis always begins in the
cytosol
• Synthesis finishes in the cytosol unless the
polypeptide signals the ribosome to attach
to the ER
• Polypeptides destined for the ER or for
secretion are marked by a signal peptide
• A signal-recognition particle (SRP) binds
to the signal peptide
• The SRP brings the signal peptide and its
ribosome to the ER
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

59.

Fig. 17-21
Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
SRP
receptor
protein
Translocation
complex
ER
membrane
Protein

60.

Point mutations can affect protein
structure and function
Mutations are changes in the genetic
material of a cell or virus
Point mutations are chemical changes in
just one base pair of a gene
The change of a single nucleotide in a
DNA template strand can lead to the
production of an abnormal protein
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings