From Genes to Proteins

Lecture objectives
• Describe how information encoded in genes
is used in the synthesis of proteins, through
– Transcription
– Translation
Central dogma of molecular biology
 Protein Synthesis
• The information contained by DNA is in the form of
specific sequences of nucleotides along the DNA
strands (Genes)
• DNA in cells dictates synthesis of proteins
• The process by which DNA directs protein synthesis
(gene expression) includes two stages, called
transcription and translation
Protein Synthesis
 Transcription and Translation
• In a eukaryotic cell the nuclear envelope separates transcription
from translation
• Extensive mRNA processing occurs in the nucleus

Nuclear
envelope

TRANSCRIPTION DNA

Pre-mRNA
RNA PROCESSING

mRNA

Ribosome

TRANSLATION

Polypeptide
 Transcription and Translation

• Cells are governed by a cellular chain of command

– DNA → mRNA → protein
• Transcription
– Is the synthesis of mRNA under the direction of DNA
– Produces messenger RNA (mRNA)
• Translation
– Is the actual synthesis of a polypeptide (protein), which
occurs under the direction of mRNA
– Occurs on ribosomes
Protein synthesis
Classes of RNA
1. Ribosomal RNA (rRNA) along with ribosomal protein subunits
make up the ribosome, the site of protein assembly.

2. Messenger RNA (mRNA) carries the coding instructions for

polypeptide chains from DNA to the ribosome and provides a
template for joining amino acids.

3. Transfer RNA (tRNA) serves as the link between the coding

sequence of nucleotides in the mRNA and the amino acid sequence
of a polypeptide chain.
• Each tRNA attaches to one particular type of amino acid and helps
to incorporate that amino acid into a polypeptide chain.
 Transcription and Translation
mRNA : Properties
• mRNA is single stranded, not double
stranded like DNA

• mRNA is short, only 1 gene long, where DNA

is very long and contains many genes

• mRNA uses the base uracil (U) instead of

thymine (T) in DNA.
Prokaryotic Transcription

• Learning Outcomes
1. Describe the transcription process in
bacteria.
2. Differentiate features of initiation from those
of elongation.
3. Define the unique features of prokaryotic
transcription
Prokaryotic Transcription
• Prokaryotes have a single RNA
polymerase which exists in two
forms: the core polymerase and
the holoenzyme.
• core polymerase
• composed of four subunits: two
identical α subunits, a β
subunit, and a β' subunit.
• synthesizes RNA using a DNA
template, The active site of the
enzyme is formed by the β and
β' subunits, which bind to the
DNA template.
• holoenzyme
• is formed by the addition of a σ
(sigma) subunit to the core
polymerase. Has the ability to
recognize specific signals in
DNA allowing RNA polymerase
to locate the beginning of
genes.
• can accurately initiate
synthesis.
 Prokaryotic Transcription:
Synthesis of an RNA Transcript
• The stages of Promoter
Transcription unit
transcription are 5 3
3 5
– Initiation Start point
DNA

– Elongation RNA polymerase 1 Initiation. After RNA polymerase binds to

the promoter, the DNA strands unwind, and
– Termination the polymerase initiates RNA synthesis at the
start point on the template strand.
Promoters signal 5
3
3
5
the initiation of Unwound RNA DNA
Template strand of

RNA synthesis. DNA transcript 2 Elongation. The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5 → 3 . In the wake of
transcription, the DNA strands re-form a double helix.
Rewound
RNA
5 3
3 3 5
5
RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.

5 3
3 5
5 3
Completed RNA
transcript
Prokaryotic Transcription:
Synthesis of an RNA Transcript- Termination

• The end of prokaryotic transcription is marked by terminator

sequences that signal “stop” to the polymerase.
• Reaching these sequences causes the formation of
phosphodiester bonds to cease, the RNA–DNA hybrid within
the transcription bubble to dissociate, the RNA polymerase
to release the DNA, and the DNA within the transcription
bubble to rewind.
 Translation
Learning outcomes
• Explain why the tRNA charging reaction is
critical to translation.
• Identify the tRNA-binding sites in the
ribosome.
• Distinguish between translation initiation and
elongation.
• Explain the elongation cycle.
Translation
Genetic code
Prokaryotic Translation
• In prokaryotes, the mRNA produced by transcription begins to
be translated before transcription is finished—i.e. they are
coupled .
• As soon as a 5' end of the mRNA becomes available,
ribosomes are loaded onto this to begin translation.
 Prokaryotic Translation
• Translation is the mRNA-directed TRANSCRIPTION DNA

synthesis of a polypeptide mRNA

Ribosome

• Translation involves
TRANSLATION
Polypeptide

– mRNA
Amino
– Ribosomes - Ribosomal RNA acids
Polypeptide
– Transfer RNA
– Codons tRNA with
amino acid
Ribosome attached

– The 5’ end of the mRNA molecule Gly

associate with ribosomes (site of
protein synthesis) tRNA

• 20 different amino acids in a cell A A A

Anticodon
that participate in protein U G G U U U G G C

synthesis 5 Codons 3
mRNA
 Translation
• Before amino acids join together TRANSCRIPTION DNA

to form a protein they attach to the mRNA

Ribosome

transfer RNA (tRNA)- a process TRANSLATION

Polypeptide

called amino acid activation

• For each different amino acid Amino
Polypeptide acids
there is a specific tRNA
tRNA with
amino acid
Ribosome attached

Gly

tRNA

Anticodon
A A A
U G G U U U G G C

5 Codons 3
mRNA
 Prokaryotic Translation
Transfer RNA
• Consists of a single RNA strand that is only about 80 nucleotides long
• Each carries a specific amino acid on one end and has an anticodon on
the other end
• A special group of enzymes pairs up the proper tRNA molecules with
their corresponding amino acids.
• tRNA brings the amino acids to the ribosomes,
3
Amino acid A
C
attachment site C
The “anticodon” is the 3 RNA bases that A
C G
5
matches the 3 bases of the codon on the G
C
C
G
mRNA molecule U G
U A
A U
U C A U
* C A C AG UA AG *
G * CUC *
C GU GU* CGAG G
* * U C * AG G
* GAG C Hydrogen
G C
U A bonds
* GA
A* C
*A U
A G
Anticodon
 Prokaryotic Translation
Transfer RNA and the charging
reaction
• Each amino acid must be attached to a
tRNA with the correct anticodon for protein
synthesis to proceed.
• This covalent attachment is accomplished
by the action of activating enzymes called
aminoacyl-tRNA synthetases.
– Cells produce 20 different aminoacyl-tRNA
synthetase enzymes, 1 for each of the 20 distinct
amino acids.
• Aminoacyl-tRNA synthetases recognizes a
specific amino acid and also ATP then
correct tRNA binds to the synthetase.
• the tRNA with its attached amino acid is
released from the enzyme. At this stage, the
tRNA is called a charged tRNA or an
aminoacyl-tRNA.
 Prokaryotic Translation
• named ribosomal RNA
Ribosomes

• Ribosome is composed of two

subunits and the interface
between the subunits is primarily
composed of rRNA.
• The bacterial ribosome contains
three binding sites,
• P site (peptidyl) binds to the
tRNA attached to the growing
peptide chain.
• A site (aminoacyl) binds to the
tRNA carrying the next amino
acid to be added.
• E site (exit) binds the tRNA that
carried the previous amino acid
added.
 Prokaryotic Translation

Building a Polypeptide
Amino end Growing polypeptide

Next amino acid

to be added to
polypeptide chain

tRNA

mRNA 3

Codons
5

(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-
pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A
site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged
tRNA leaves via the E site.
 Prokaryotic Translation
Building a Polypeptide

• We can divide translation into three stages

– Initiation
– Elongation
– Termination
• The AUG start codon is recognized by methionyl-tRNA
• Once the start codon has been identified, the ribosome
incorporates amino acids into a polypeptide chain
• RNA is decoded by tRNA (transfer RNA) molecules, which each
transport specific amino acids to the growing chain
• Translation ends when a stop codon (UAA, UAG, UGA) is reached
 Prokaryotic Translation
Initiation of Translation
• The initiation stage of translation brings together mRNA, tRNA
bearing the first amino acid of the polypeptide, and two subunits
of a ribosome
Large
ribosomal
P site subunit
3 U A C 5
5 A U G 3

Initiator tRNA
GTP GDP
E A
mRNA
5 3 5 3
Start codon

mRNA binding site Small Translation initiation complex

ribosomal
subunit

1 2
A small ribosomal subunit binds to a molecule of
mRNA. In a prokaryotic cell, the mRNA binding site
on this subunit recognizes a specific nucleotide
sequence on the mRNA just upstream of the start
codon. An initiator tRNA, with the anticodon UAC,
base-pairs with the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
The arrival of a large ribosomal subunit completes
the initiation complex. Proteins called initiation
factors (not shown) are required to bring all the
translation components together. GTP provides
the energy for the assembly. The initiator tRNA is
in the P site; the A site is available to the tRNA
bearing the next amino acid.
•
Prokaryotic Translation
In the elongation stage, amino acids are added one by one to
the preceding amino acid
Elongation of the Polypeptide Chain
1 Codon recognition. The anticodon
TRANSCRIPTION DNA
Amino end of an incoming aminoacyl tRNA
mRNA
of polypeptide base-pairs with the complementary
Ribosome
TRANSLATION mRNA codon in the A site. Hydrolysis
Polypeptide
of GTP increases the accuracy and
E efficiency of this step.
mRNA 3
Ribosome ready for P A
next aminoacyl tRNA 5 site site
2 GTP
2 GDP

E E

P A P A

2 Peptide bond formation. An

GDP
3 Translocation. The ribosome rRNA molecule of the large
GTP
subunit catalyzes the formation
translocates the tRNA in the A
of a peptide bond between the
site to the P site. The empty tRNA
new amino acid in the A site and
in the P site is moved to the E site, E
the carboxyl end of the growing
where it is released. The mRNA
polypeptide in the P site. This step
moves along with its bound tRNAs,
P A attaches the polypeptide to the
bringing the next codon to be
tRNA in the A site.
translated into the A site.
 Prokaryotic Translation
Termination of Translation
• The final stage is termination when the ribosome reaches a stop codon
in the mRNA

Release
factor
Free
polypeptide

5
3 3
3
5 5
Stop codon
(UAG, UAA, or UGA)
1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal subunits
codon on mRNA, the A site of the the bond between the tRNA in and the other components of
ribosome accepts a protein called the P site and the last amino the assembly dissociate.
a release factor instead of tRNA. acid of the polypeptide chain.
The polypeptide is thus freed
from the ribosome.
Eukaryotic Transcription

Learning Outcomes
• List the different eukaryotic RNA polymerases.
• Distinguish between the promoters of the RNA
polymerases.
• Define the processing that occurs to eukaryotic
transcripts.
Eukaryotic Transcription

• eukaryotes have three different RNA polymerases,

distinguished in both structure and function.
• i.e.
1. RNA polymerase I transcribes rRNA,
2. RNA polymerase II transcribes mRNA and some small
nuclear RNAs,
3. RNA polymerase III transcribes tRNA and some other small
RNAs.
• Together, these three enzymes accomplish all
transcription in the nucleus of eukaryotic cells.
• Each polymerase has its own promoter.
Eukaryotic Transcription
• Transcription of eukaryotic structural genes is
initiated when RNA polymerase II and general
transcription factors bind to a promoter
sequence.
Eukaryotic Transcription
• Transcriptional termination of RNA polymerase II occurs
after the 3ʹ end of the transcript is cleaved near the polyA
signal sequence.
• After RNA polymerase II has transcribed the polyA signal
sequence, the RNA is cleaved just downstream from this
sequence.
• Cleavage occurs before transcriptional termination.
• Two models account for transcription termination i.e.
1. RNA polymerase II becomes destabilized after it has
transcribed the polyA signal sequence, and it eventually
dissociates from the DNA.
2. RNA polymerase II is physically removed from the DNA.
Region of RNA that is downstream from the polyA signal
sequence is cleaved by an exonuclease that degrades the
transcript in the 5ʹto 3ʹ direction. When the exonuclease
catches up to RNA polymerase II, this causes RNA
polymerase II to dissociate from the DNA.
Eukaryotic Transcription
Post-transcriptional modification
• Eukaryotic mRNA molecules are modified in the nucleus with
the addition of a methylated GTP to the 5' end of the
transcript, called the 5' cap, and a long chain of adenine
residues to the 3' end of the transcript, called the 3'poly-A tail.
• The 5' cap protects the 5' end of the mRNA
from degradation and is also involved in
translation initiation
• The 3' poly-A tail appears to play a role in
• the stability of mRNAs by protecting them
from degradation.
Eukaryotic Transcription
Pre-mRNA splicing
• Eukaryotic genes contain sequences that form the coding
sequence called exons and intervening sequences called
introns.
• Removal of introns is called pre-mRNA splicing, is
accomplished by an organelle called the spliceosome.
Eukaryotic Transcription
Pre-mRNA splicing
• Pre-mRNA splicing occurs
in the nucleus prior to the
export of the mRNA to the
cytoplasm.
• The intron–exon junctions
are recognized by small
nuclear ribonucleoprotein
particles, called snRNPs
(pronounced “snurps”).
• snRNPs are complexes
composed of snRNA and
protein which cluster
together with other
associated proteins to form
a larger complex called the
spliceosome, which is
responsible for the splicing,
or removal, of the introns.
 Study break
• Describe the structure and function of a spliceosome.
• In eukaryotes, what types of modification occur to pre-
mRNA.
Eukaryotic Translation
Learning outcomes
1. Distinguish between translation initiation and elongation.
2. Explain the elongation cycle.
Eukaryotic Translation
• Eukaryotic initiation the initiating amino acid is methionine
 Eukaryotic Translation

• Elongation continues in this fashion until a chain-terminating stop codon is reached.

• These stop codons do not bind to tRNA; instead, they are recognized by release factors,
proteins that release the newly made polypeptide from the ribosome
 Study break
A template strand of DNA has the following sequence:
3' – CGTTACCCGAGCCGTACGATTAGG – 5'
Use the sequence information to determine
a. the predicted sequence of the mRNA for this gene.
b. the predicted amino acid sequence of the protein.
 DNA
TRANSCRIPTION

3

RNA
5 RNA polymerase
transcript
RNA PROCESSING
Exon
Summary of RNA transcript
transcription (pre-mRNA)

and translation Intron

in a eukaryotic Aminoacyl-tRNA
synthetase
cell NUCLEUS

Amino
acid AMINO ACID ACTIVATION
CYTOPLASM tRNA

mRNA Growing
polypeptide
3
A
Activated
P amino acid
Ribosomal
E
subunits

5
TRANSLATION

E A
Anticodon

Codon

Ribosome