Chapter 17

From Gene to Protein

 Overview: The Flow of Genetic Information
• the information content of DNA is in the form of specific sequences
of nucleotides
• the DNA inherited by an organism leads to specific traits by
dictating the synthesis of proteins
• proteins are the links between genotype and phenotype
• Gene expression = process by which DNA directs protein synthesis
– includes two stages: transcription and translation
Basic Principles of Transcription and Translation

• RNA is the bridge between genes and the proteins for which they
code
• Transcription = synthesis of RNA
– using information in DNA
• Translation = synthesis of a polypeptide
– using information in the mRNA
– Ribosomes - sites of translation

DNA RNA Protein

The Products of Gene Expression: A Developing Story

• original hypothesis posed by scientists: one gene – one

enzyme
• BUT a lot of proteins aren’t enzymes - researchers later
revised the hypothesis: one gene–one protein
• many proteins are composed of several polypeptides
– each of which has its own gene
• can now restated the hypothesis as the one gene–one
polypeptide hypothesis
• **Note: common to refer to gene products as proteins rather
than polypeptides
DNA vs. RNA
 Types of RNA
 mRNA = messenger RNA
– majority of RNA found in a cell
– carries the genetic information which will be translated into a
protein sequence
– defined by the presence of a “cap” at its 5’ end and a long tail of
adenines at its 3’ end = “poly-A tail”
 Types of RNA

 rRNA = ribosomal RNA

 found in the nucleolus
 combines together with the large and small ribosomal subunits to
form the functional ribosome (protein translation)
 rRNA is transcribed in the nucleolus by RNA polymerase I

28S rRNA
 Types of RNA
 tRNA = transfer RNA
 actually translates the message coded in the mRNA into a protein
sequence which will become a function protein
 tRNA is transcribed in the nucleoplasm by an enzyme called RNA
polymerase III
 then exported into the cytoplasm where AA are added
 5’ 3’

3’ 5’

-transcription of RNA is similar to DNA replication – RNA is made in the 5’ to 3’

direction
-enzyme called an RNA polymerase binds to only one of the DNA strands = the
anti-sense (template strand)
-it moves along the template DNA strand (in the 3’ to 5’ direction) and reads the
nucleotide and adds a complementary RNA base
- a growing strand of RNA complementary to the DNA strand results
-BUT rather than a T being paired with an A – U becomes the partner to A
 Transcription
-a human gene is also known as a transcription unit = stretch of DNA that is transcribed
into RNA
-a transcription units is comprised of:
1. coding sequence – gives rise to protein strand upon translation
-contains regions of code = “exons” – code for amino acids
-and regions of junk = “introns” – spliced out in the nucleus

3’
5’ Exon Intron Exon Intron Exon Intron Exon
 Transcription
- 2. untranslated regions (UTRs) - the regions upstream and downstream of the
coding region that are transcribed but NOT translated into a protein
- -play an important role in translation – can influence the binding of the ribosome
to the mRNA
- -also play a role in exporting the mRNA into the cytoplasm
 Transcription
• genes are also associated with additional sequences of DNA
1. core promoter sequence – for the binding of the RNA polymerase
-RNA polymerase recognizes specific sequences of nt’s
-binding is helped out by transcription factors
2. enhancer regions – help enhance transcription
can be several thousands of base pairs upstream of the gene
 Transcription
• the transcription unit is transcribed by an RNA polymerase
• three types of RNA polymerase – I, II and III
• RNA polymerases create an RNA strand called a primary transcript
• must be modified to produce the final mRNA, tRNA or rRNA
• RNA polymerase II transcribes protein coding genes into a primary transcript called pre-
mRNA – this is then is processed into mRNA
– genes for tRNA are transcribed in the cytoplasm by RNA polymerase III – primary
transcript is modified into tRNA
– genes for rRNA is transcribed in the nucleolus by RNA polymerase I – primary
transcript is modified into rRNA

-3D representation of the

RNA polymerase II enzyme
 Transcription
• three stages of transcription
– Initiation: binding of the RNA polymerase to the
promoter
• special sequences denote this region
– Elongation: movement of the RNA polymerase along
the anti-sense DNA strand and synthesis of the RNA
transcript
– Termination: release of the RNA polymerase from
the DNA
• special sequences denote this region
• differs between prokaryotes and eukaryotes
 Promoter Transcription unit

5 3
3 5
DNA
Start point
RNA polymerase 1. Initiation – RNA polymerase binds to a special sequence
of nucleotides called the promoter

-certain sections of the promoter are important in 1 A eukaryotic promoter

polymerase binding = core promoter Promoter
Nontemplate strand
DNA
-in prokaryotes the promoter binds the RNA 5 TAT AAAA
AT AT T T T
3
3 5
polymerase without help TATA box Start point Template strand

-in eukaryotes – the polymerase requires the assistance Transcription

2 Several transcription
factors bind to DNA
of proteins called transcription factors factors

-specific transcription factors bind to the promoter 5 3

first and then help position the polymerase at the 3 5

promoter 3 Transcription initiation

complex forms
-additional transcription factors then bind RNA polymerase II
Transcription factors
-entire complex is called the Transcription Initiation
Complex 5
3 5 3 3
5

RNA transcript

Transcription initiation complex

sequence given in texts is that of the sense strand

 Promoter Transcription unit

5 3
3 5
DNA
Start point
RNA polymerase 1 Initiation

Nontemplate strand of DNA

5 3
3 5
Template strand of DNA
RNA
Unwound
transcript
DNA

1. Initiation cont…
-RNA polymerase unwinds the DNA helix (acts as a helicase) –
exposes about 10 to 20 nucleotides for copying
-RNA polymerase holds the DNA helix open (acts like the SSBs)
-RNA polymerase initiates RNA synthesis without the need for a
primer
 Promoter Transcription unit 2. Elongation – RNA polymerase synthesizes
a complementary RNA strand
5 3 -RNA primary transcript grows in the 5’ to 3’
3 5
DNA direction
Start point
RNA polymerase -uses uracil instead of thymine
1 Initiation
-the DNA strands reform their helix once the
RNA polymerase moves past the area
Nontemplate strand of DNA -the mRNA strand emerges from the
5 3
3 5 polymerase-DNA complex
Template strand of DNA
RNA
Unwound
transcript
DNA
2 Elongation

Rewound
DNA Nontemplate
strand of DNA
5 3 RNA nucleotides
3 3 5 RNA
5 polymerase

RNA 3
A T C C A A 5
transcript C 3 end

C A U C C A
5 3
T A G G T T

Multiple RNA polymerases per DNA template 5 Direction of transcription

Template
strand of DNA
Newly made
RNA
 Promoter Transcription unit

5 3 3. Termination – RNA polymerase

3 5
DNA reaches a specific sequence of
Start point
RNA polymerase nucleotides and stops
1 Initiation
transcription
-the RNA polymerase detaches
Nontemplate strand of DNA from the DNA
5 3
3 5 -the pre-RNA primary transcript is
RNA
Template strand of DNA released
Unwound
transcript
DNA
2 Elongation -in prokaryotes – a termination
Rewound sequence that detaches the
DNA polymerase
5
3
3 -in eukaryotes – the RNA
3 5
5 polymerase transcribes a
RNA
sequence called a poly-
transcript 3 Termination adenylation signal
– for the release of the pre-RNA
5 3 from the polymerase
3 5
5 3
Completed RNA transcript

Direction of transcription (“downstream”)

 Transcription
• to modify the primary transcript into mRNA – the
following modifications are made:
– a 5’methylated cap is added to the 5’end
– addition of a 3’ poly A tail
– the coding sequence is “edited” = splicing
Eukaryotic cells modify RNA after transcription

• enzymes in the eukaryotic nucleus modify pre-mRNA before

exporting the mRNA to the cytoplasm
– known as RNA processing
• 5’ methylated cap – plays a role in the docking of the ribosome
to mRNA – for translation
– modified guanine nucleotide added after the transcription of about 20 to 40
nucleotides

Protein-coding Polyadenylation
segment signal
5 3
G P P P AAUAAA AAA … AAA

Start Stop
5 Cap 5 UTR 3 UTR Poly-A tail
codon codon
Eukaryotic cells modify RNA after transcription
• 3’ poly A tail – plays a role in the export of the mRNA
into the cytoplasm
– after transcription – an enzyme adds 20 to 250 adenine nucleotides after
the poly-adenylation signal sequence
– also prevents degradation of the mRNA once its in the cytoplasm

Protein-coding Polyadenylation
segment signal
5 3
G P P P AAUAAA AAA … AAA

Start Stop
5 Cap 5 UTR 3 UTR Poly-A tail
codon codon
 RNA Splicing
• most eukaryotic genes and pre-RNA transcripts have long noncoding stretches of
nucleotides that lie between coding regions
– the noncoding regions are called intervening sequences, or introns
– coding regions are called exons because they are eventually expressed in the form of a
protein
– RNA splicing removes introns and joins exons, creating an mRNA molecule
with a continuous coding sequence
– the way you splice can also create multiple isoforms from one RNA transcript

5 Exon Intron Exon Intron Exon 3

Pre-mRNA 5 Cap Poly-A tail
Codon 130 31104 105
numbers
146
Introns cut out and
exons spliced together

mRNA 5 Cap Poly-A tail

1146
5 UTR 3 UTR
Coding
segment
• RNA splicing is carried out by spliceosomes
• Spliceosomes = several proteins and small nuclear
ribonucleoproteins (snRNPs) that recognize specific sequences
found in introns called splice sites
• snRNPs – found in the nucleus and are made of small nuclear RNA
(snRNA) and proteins

RNA transcript (pre-mRNA)

5
Exon 1 Intron Exon 2
Protein
Other
snRNA proteins
snRNPs
 RNA transcript (pre-mRNA)
5
Exon 1 Intron Exon 2
Protein
Other
snRNA proteins
snRNPs
1. snRNPs and other proteins
Spliceosome
combine to form the spliceosome

5
2. the spliceosome brings the ends
of two exons together
-forms a “lariat” out of the intron

Spliceosome
components
Cut-out
3. the spliceosome cuts the mRNA intron
pre-mRNA and releases the intron 5
for degradation Exon 1 Exon 2
 RNA Splicing
• genes can encode for more than one Gene
protein DNA
– depending on what segments of RNA are Exon 1 Intron Exon 2 Intron Exon 3
treated as exons and what are treated as
introns during splicing Transcription

• so the way you splice can determine

what proteins eventually get made = RNA processing

alternative RNA splicing

Translation
• proteins often are composed of discrete
regions called domains – coded for by
Domain 3
distinct exons
– cut out a domain – get a different protein
• also - exon shuffling may result in the Domain 2

evolution of new proteins Domain 1

– introns increase the probability of crossing-
over between alleles Polypeptide
– creates new exon combinations
 Splicing
• for an animation go to
http://sumanasinc.com/webcontent/animatio
ns/content/mRNAsplicing.html
• (don’t worry about the actual proteins!)
 Translation
• process of converting an mRNA message into a strand of amino acids that will be
processed into a mature functional protein
• performed by the ribosome in combination with tRNA molecules
• prokaryotes - translation of mRNA can begin before transcription has finished – no
separation between the mRNA and the ribosome
• eukaryotic cell- the nuclear envelope separates transcription from translation
– mRNA has to be exported out of the nucleus first

DNA
template 3 5 DNA
strand A C C A A A C C G A G T molecule

T G G T T T G G C T C A
Gene 1
5 3
TRANSCRIPTION
Gene 2
U G G U U U G G C U C A
mRNA 5 3
Codon
TRANSLATION

Protein Trp Phe Gly Ser

Gene 3
Amino acid
• How are the instructions for assembling
amino acids into proteins encoded into The Genetic Code
DNA?
1964
• 20 amino acids - only four nucleotide bases
in DNA Second mRNA base

• how many nucleotides correspond to an U C A G

UUU UCU UAU UGU U
amino acid? Phe Tyr Cys
UUC UCC UAC UGC C
• the mRNA nucleotide sequence is “read” in U Ser
UUA UCA UGA Stop A
groups of 3 nucleotides = “codons” Leu
UAA Stop

• each codon codes for 1 of the 20 amino UUG UCG UAG Stop UGG Trp G

acids that make up proteins CUU CCU CAU CGU U

His
• called the “genetic code”

Third mRNA base (3 end of codon)

First mRNA base (5 end of codon)
CUC CCC CAC CGC C
C Leu Pro Arg
– 61 amino acid codons; 3 stop codons CUA CCA CAA CGA A
• the code is redundant - each amino acid CUG CCG CAG
Gln
CGG G
can be coded for by more than one codon
• e.g. alanine – GCU, GCC, GCA and GCG AUU ACU AAU
Asn
AGU
Ser
U
• the GC defines the amino acid as alanine A
AUC Ile ACC AAC AGC C
Thr
• in many cases the 3rd codon is important in AUA ACA AAA
Lys
AGA
Arg
A
defining the amino acid AUG Met or
start ACG AAG AGG G
– serine –codons are: AGU, AGC
GUU GCU GAU GGU U
– BUT arginine codons are: AGA and AGG Asp
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G
Molecular Components of Translation

• two components
• 1. transfer RNA (tRNA)
• 2. the ribosome
tRNA
• tRNA molecule consists of a single RNA strand that is only about 80 nucleotides
long
• at one end – anticodon site for the hybridization with the mRNA template
• at the other end – attachment site for the amino acid that corresponds to the
mRNA codon
• transcribed in the cytoplasm by RNA polymerase III – it folds into its
characteristic shape spontaneously due
3
to regions that complement each other
Amino acid
attachment
site 5
Amino acid
attachment
5 site
3

Hydrogen
bonds
Hydrogen
bonds

A A G
3 5
Anticodon Anticodon
Anticodon
(c) Symbol used
(a) Two-dimensional structure (b) Three-dimensional structure in this book
 Aminoacyl-tRNA
synthetase (enzyme)

Amino acid

P Adenosine

P P P Adenosine P Pi

ATP Aminoacyl-tRNA
Pi
Pi tRNA synthetase

-amino acids are attached in tRNA

the cytoplasm by enzymes
called Amino
aminoacyl-tRNA –synthetases acid
-one end fits the amino acid,
the other end fits the tRNA P Adenosine

-20 synthetases – each is specific AMP

for only one kind of tRNA Computer model
-the tRNA attached to an AA is
called a ‘charged tRNA’

Aminoacyl tRNA
(“charged tRNA”)
tRNA and the 3rd codon “wobble”
• the tRNA recognizes the codon “triplet” on the mRNA
template
• attached to the tRNA is the amino acid corresponding
to this codon
• there are 61 amino acid codons – so there should be
61 tRNAs
• there are only 45 tRNAs
– some tRNAs can bind more than one codon
• the rules for complementary base pairing at the third
NT of the codon are less stringent
– “flexible” base pairing at this NT = Third Codon Wobble
Ribosomes
• machine of translation
• made in the nucleolus in eukaryotic cells
• comprised of two subunits of proteins (large and small) linked
together with a piece of rRNA
– eukaryotes: 40S small subunit = 33 proteins + 18S rRNA
+ 60S large subunit = 50 proteins + 28S rRNA (+ 5.6S rRNA + 5S rRNA)
– rRNA is transcribed in the nucleolus, proteins are imported from cytoplasm
– everything is assembled in the nucleolus
– subunits are exported out via nuclear pores
– prokaryotic ribosomes and similar but smaller
Ribosomes
• within the large subunit are two sites for the binding of tRNA
– P-site or Peptidyl-tRNA site – “old” AA
– A-site or aminoacyl-tRNA site – incoming AA
• and one E site/Exit site for the exit of the tRNA off the
ribosome
P site (Peptidyl-tRNA
Exit tunnel
binding site)

A site (Aminoacyl-
tRNA binding site)
E site
(Exit site)

E P A Large
subunit

mRNA
binding site Small
subunit
Ribosomes
• eukaryotic ribosomes are similar but are larger vs. prokaryotes
• most evidence now identifies the rRNA as being the catalyst for
the formation of the peptide bond and the growth of the
polypeptide chain
– RNA with enzymatic activity = ribozyme

Growing polypeptide
Amino end
Next amino
acid to be
added to
polypeptide
chain

E tRNA
mRNA 3

5 Codons

(c) Schematic model with mRNA and tRNA

 Building a Polypeptide
• 3 stages of translation:
– Initiation
– Elongation
– Termination
• all three stages require protein “factors”
– called initiation factors or IFs
– in eukaryotes – known as eIFs
 1. Initiation of Translation
• the small subunit of the ribosome binds onto the mRNA sequence near the 5’ methylated
cap
• this subunit already has an initiator tRNA (bound to methionine) associated with it
• binding of the small subunit is helped by numerous eukaryotic initiation factors (eIFs)
• the small subunit then glides down the mRNA “scanning” for the first codon - START codon
= AUG (methionine)
-stops so that initiator tRNA can hybridize with the start codon

Large
ribosomal
subunit
3 U A C 5 P site
5 A U G 3
P i
Initiator 
tRNA GTP GDP
E A
mRNA
5 5
Start codon
3 3
Small
mRNA binding site ribosomal Translation initiation complex
subunit
• once the small subunit is positioned - the large subunit then
assembles and completes the ribosomal “machine”
• helped by even more eIF’s
• the mRNA and the ribsosome form the Translation Initiation
Complex
• the eIF’s are released once this complex forms
• the ribosome is now ready for the next AA - elongation
follows
Large
ribosomal
subunit
3 U A C 5 P site
5 A U G 3
P i
Initiator 
tRNA GTP GDP
E A
mRNA
5 5
Start codon
3 3
Small
mRNA binding site ribosomal Translation initiation complex
subunit
2. Elongation
of Translation
http://www.youtube.com/watc
h?v=5bLEDd-PSTQ
http://www.youtube.com/watc
h?v=Ikq9AcBcohA
http://www.youtube.com/watc
h?v=NJxobgkPEAo
2. Elongation
of Translation
3. Termination of Translation
Release
factor Free
polypeptide

5
3 3
2 GTP 3
5 5
2 GDP  2 P
Stop codon
(UAG, UAA, or UGA)

-translation also stops at specific codons = STOP codons

-UAA, UGA, UAG
-so when the ribosome reaches these sequences – no more AAs are added and the
ribosome detaches from the peptide strand and mRNA
-a release factor cleaves the polypeptide chain from the tRNA and releases it from the
ribosome (GTP hydrolysis)
-the translation machine “breaks apart” – requires an enzyme that uses ATP hydrolysis
 Polyribosomes
• a number of ribosomes can Completed
polypeptide
translate a single mRNA Growing
polypeptides
simultaneously, forming a
polyribosome (or polysome) Incoming
ribosomal
• polyribosomes enable a cell to subunits

make many copies of a

polypeptide very quickly Start of
mRNA End of
(5 end) mRNA
(3 end)
(a)

Ribosomes

mRNA

(b)
0.1 m