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Kim H Brown, PhD
1. Relating proteins (traits) to genetic material
2. Structure of Proteins
3. The central dogma of biology: Eukaryotes vs
a) Transcription
b) RNA processing
c) Translation
d) The Ribosomal Machinery
e) The Genetic Code

TED Talk “The astonishing molecular machines” (3:13-6:00:
Simple gene expression: http://highered.mcgraw-
Gene Expression Steps
The principal steps in gene expression are:
• Transcription: RNA molecules are
synthesized from a DNA template
• Processing (eukaryotic): RNA undergoes
chemical modification
• Translation: RNA is the blueprint for a
• The protein made is called the gene
product, and is a consequence of the
base sequence in the DNA
Proteins are essential to the structures and
functions of life
A protein is a polymer built from various combinations of 20
amino acid monomers
Amino acids: have an amino group and a carboxyl group
– covalently bonded to a central carbon atom
– central carbon also bonded to hydrogen atom and another
chemical group symbolized by R
Polypeptides (proteins) are made from amino
acids linked by peptide bonds
Polymerization of proteins: enzyme-mediated dehydration
– Peptide bond: Carboxyl group of one amino acid linked
to amino group of the next amino acid
– The sequence of amino acids in proteins is specified by
the coding information in specific genes
Colinearity: the sequence of base pairs in DNA determines the
sequence of amino acids in the polypeptide in a colinear, or
point-to-point, manner
• Colinearity is universally found in prokaryotes, whereas In
eukaryotes, noninformational DNA sequences interrupt the
continuity of most genes

Colinearity of DNA and protein in the trpA gene of E. coli.

A protein’s shape depends on four levels of
• The amino acid sequence causes
the polypeptide to assume a
particular shape
– The shape of a protein
determines its specific function
• If for some reason a protein’s
shape is altered, it can no longer
– Denaturation will cause
polypeptide chains to unravel
and lose their shape and, thus,
their function

Crash Course Prions Disease:
Amino acids are classified as hydrophobic or
– Some amino acids have a nonpolar R group and are
– Others have a polar R group and are hydrophilic, which
means they easily dissolve in aqueous solutions
Protein Domains
Protein Domains: regions fold
into well-defined structures often
have specialized functions
• Individual domains: usually have
independent evolutionary origins
• come together in various
combinations via duplication
and genomic rearrangements
• Can be identified through
computer analysis of the amino
acid sequence

Pyruvate kinase, a protein with three domains.
Transcription is the
process of synthesis of an
RNA molecule copied from
the segment of DNA that
constitutes the gene

Unlike DNA, RNA

1. Is single stranded
2. Has ribose sugar
instead of RNA synthesis: http://highered.mcgraw-
deoxyribose r3/mrna_synthesis__transcription_.html
3. Has uracil in place of
RNA Synthesis
In the synthesis of RNA, a sugar–phosphate bond is formed
between the 3'- hydroxyl group of one nucleotide and the 5'- OH
triphosphate of the next nucleotide in line
1. The nucleotide sequence in the transcribed mRNA is
complementary to the base sequence in DNA
2. RNA synthesis does not require a primer
3. The enzyme used in transcription is RNA polymerase
RNA Polymerases
• RNA polymerases are large,
multisubunit complexes
whose active form is called
the RNA polymerase
• Bacterial cells have only
one RNA polymerase
holoenzyme, which
contains six polypeptide

Bacterial transcription: http://highered.mcgraw-
RNA Synthesis
Promoter: nucleotide sequence, 20–200 bp long, is the initial
binding site of RNA polymerase and transcription initiation factors
• Promoter recognition by RNA polymerase is a prerequisite for
transcription initiation
The consensus promoter regions in E. coli are:
TTGACA (-35), centered approximately 35 base pairs upstream
from the transcription start site (numbered the +1 site)
TATAAT (-10) = “TATA”-like box: 10 base pairs upstream

Base sequences in promoter regions of several genes in E. coli.

RNA Synthesis

Transcription termination sites are

inverted repeat
sequences that can form loops in
RNA: stop signal

Stages of transcription:

Eukaryotic Transcription
• First, a primary transcript is formed from DNA template
• Primary transcript is processed to form mRNA that is
transported to the cytoplasm

Control of Eukaryotic gene expression:

RNA Polymerases
Eukaryotes have several types of RNA polymerase:
• RNA polymerase I transcribes ribosomal RNA.
• RNA polymerase II–all protein-coding genes as well as the
genes for small nuclear RNAs
• RNA polymerase III–tRNA genes and the 5S component
of rRNA
The Eukaryotic Promoter


Eukaryotic Transcription
• Initiation of protein synthesis may begin many nucleotides
downstream from the 5’-end
• The 5’ untranslated region is followed by an open reading
frame (ORF), which specifies polypeptide chain
• In many eukaryotic genes ORFs are interrupted by noncoding
segments, introns
• Primary transcript contains exons and introns; introns are
subsequently removed by splicing
Eukaryotic Transcription
• The first processing step adds 7-methylguanosine cap to the
5'-end of the primary transcript,
• The 3'-end of an mRNA molecule following the ORF also is not
translated; it is called the 3’ untranslated region
• The 3'- end is usually modified by the addition of a sequence
called the poly-A tail
• RNA splicing occurs in nuclear particles known as
• The specificity of splicing comes from the five small
snRNP—RNAs denoted U1, U2, U4, U5, and U6, which
contain sequences complementary to the splice junctions

How Spliceosomes Process RNA

Snrnps and splicesome assembly

RNA Splicing:
• The average human gene
occupies 27 kb of genomic DNA,
yet only 1.3 kb (~ 5 %) is used to
encode amino acids
• The correlation between exons
and domains found in some
genes suggests that the genes
were originally assembled from
smaller pieces
• The model of protein evolution
through the combination of
different exons is called the exon
shuffle model
• The mRNA is translated in the 5' -to-3' direction. The
polypeptide is synthesized from the amino end toward the
carboxyl end
The translation system consists
of five major components:
1. Messenger RNA: coding
sequence of bases
2. Ribosomes on which
protein synthesis takes
3. Transfer RNA translates
codons into amino acid
4. Aminoacyl-tRNA
synthetases: catalyzes
attachment of an amino
acid to its corresponding
tRNA molecule
5. Initiation, elongation, How Translation Works: http://highered.mcgraw-
and termination factors w_translation_works.html
Translation: Initiation
In eukaryotes, initiation takes
place by scanning the mRNA for
an initiation codon (AUG)
1. eIF4F binds to the 5' cap on
the mRNA and recruits eIF4A
and eIF4B
2. This creates a binding site for
a charged tRNAMet (an initiator
tRNA), bound with eIF2, and a
small 40S ribosomal subunit
together with eIF3 and eIF5
3. These components all come
together at the 5' cap and form
the 48S initiation complex
Translation: Initiation
4. Initiation complex moves along
mRNA in 3' direction, scanning for
the first AUG
5. eIF5 causes release of initiation
factors and recruitment of a large
60S ribosomal subunit
6. This subunit includes binding sites
for tRNA molecules: the E (exit)
site, the P (peptidyl) site, and the
A (aminoacyl) site.
7. tRNAMet is located in the P site
and the A site is the next in line to
be occupied.
8. tRNA binding is accomplished by
hydrogen bonding between the
AUG codon in the mRNA and the
three-base anticodon in the tRNA.
Translation: Elongation
1. The first step: the 40S subunit moves one codon farther
along the mRNA, and the charged tRNA corresponding to the
new codon is brought into the A site on the 60S subunit
2. A peptidyl transferase catalyzes a reaction in which the
bond connecting the methionine to the tRNAMet is transferred
to the amino group of the next amino acid, forming the first
peptide bond
Translation: Elongation
3. The next step: the 60S subunit swings forward to catch
up with the 40S, and simultaneously the tRNAs in the P
and A sites of the large subunit are shifted to the E and P
sites, respectively
Translation: Elongation
4. One cycle of elongation is now completed, and the entire
procedure is repeated for the next codon.
• Eukaryotes synthesize a polypeptide chain at the rate of
about 15 amino acids per second
• Elongation in prokaryotes is a little faster (about 20 amino
acids per second), but the essential processes are very
Translation: Termination
• When a stop codon is
encountered, the tRNA holding
the polypeptide remains in the
P site, and a release factor
(RF) binds with the ribosome.
• GTP hydrolysis provides the
energy to cleave the
polypeptide from the tRNA to
which it is attached
• The 40S and 60S subunits are
recycled to initiate translation
of another mRNA
• Most polypeptides fold
correctly upon exiting
ribosome: they pass through
a tunnel in large ribosomal
subunit that is 35 amino acids
• Emerging from tunnel, protein
enters into a protein cradle
where it folds
• The proper folding of more
complex polypeptides is
aided by proteins called
chaperones and
Translation: Prokaryotes
1. In prokaryotes, mRNA molecules have no cap and there is no
scanning mechanism
2. In E. coli, IF-1 and IF-3 initiation factors interact with the 30S
subunit and IF-2 binds with a special tRNA charged with
formylmethionine tRNAfMet
3. These components bind with an mRNA at the ribosome-
binding site, RBS or the Shine–Dalgarno sequence.
Together, they recruit a 50S sub-unit
4. mRNA molecules contain information for the amino acid
sequences of several different proteins; such a molecule is
called a polycistronic mRNA
5. Cistron: DNA sequence that encodes a single polypeptide
6. In a polycistronic mRNA, each protein coding region is
preceded by its own ribosome-binding site and AUG initiation
codon Initiation: http://highered.mcgraw-
Translation: Prokaryotes
• After the synthesis of one polypeptide is finished, the next
along the way is translated
• The genes contained in a polycistronic mRNA often encode
the different proteins of a metabolic pathway.

Elongation: http://highered.mcgraw-

Termination: http://highered.mcgraw-
Genetic Code
The genetic code is the list of all
codons and the amino acids that
they encode
1. Translation starts from a
fixed point and a single
reading frame maintained
throughout the process of
2. Genetic code is universal: the same triplet codons specify
the same amino acids in all species
3. Each codon consists of three nucleotides.
4. Code is non-overlapping
5. Code is degenerate: each amino acid is specified by more
than one codon
• Mutations occur when changes in codons alter amino
acids in proteins
• Mutations that delete or add a base pair shift the reading
frame and are called frameshift mutations.
How Is Gene Expression Regulated?
1. Cells can control the
frequency at which an
individual gene is transcribed
2. The same gene may be used
to produce different mRNAs
and protein products
3. Cells may control the stability
and translation of messenger
4. Proteins may require
modification before they can
carry out their functions
5. Cells can control the rate at
which proteins are degraded
1. Certain enzymes in a cell
add methyl groups (CH3)
to cytosine bases during a
process called methylation

2. Methyl groups affect the

rate of transcription without
affecting the DNA
sequence by changing the
shape of the chromosome