Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 1
Protein Synthesis in eukaryotes
Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:Trety001@umn.edu"» - 6151
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof. e-mail: :ariska@latnet.lv
Required reading: Stryer 4th Ed. Ch. 34 p. 903-906
Eukaryotic Ribosome
Ribosome
60S
40S 
Subunit Subunit



80S
Eukaryotes
60S subunit
40S subunit
28S RNA
5S RNA
5.8S RNA
18S RNA

60S
28S RNA + 5S RNA
+5.8S RNA
+
 34 proteins + multiple proteins
40S 21 proteins + multiple proteins
60S


18S RNA
Prokaryotes
50 S
30S
23S RNA
5S RNA
16S RNA
Table 1. The genetic code. for mRNA 
1st position
(5' end) 
U
C
A
G
Genetic Code
2nd position
U
C
A
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met init
Val
Val
Val
Val
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
Tyr
Tyr
STOP
STOP
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
G
Cys
Cys
STOP SelenoCys Trp Mitochondria
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
3rd position
(3' end) 
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of
protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into
valine and glutamic acid, respectively. Note that those codons with U or C as the second 2 nucleotide tend to
specify the more hydrophobic amino acids AA.
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 2
Initiation of Translation: Ribosomal Binding Site
Shine-Delgarno
16S
U CCUCCA
mRNA 5’ GA U U CC U A GG AGG U U U GACC U A U G GCC U U U 3’
Protein
fMet Ala
Phe
• Initiating AA tRNA is Met, not fMet;
• Initiator tRNA is Met-tRNA;
• Initiatior signal is AUG – no Shine-Delgarno sequence;
• 40S subunit-Met-tRNA; - initiation factors complex binds 5’ cap and scans mRNA for the nearest A U G
H
H
Caps 0,1,2
O
O
P
O
O
O
H O
H
H
N
N
N
N
H
O
O
O
+
N
H
H
H
O P O P O
O
H
A or
G
O
H
O H
H
O
O
O
P
O
H
Caps 1 and 2
H
O
H
H
H
O
Eukaryotic mRNA is 5’-Capped
Base
O
O
H
Cap 2
H
Eukaryotic counterparts of bacterial factors
Initiation
Bacteria
IF 1, 3
IF 2
-
Eukaryotes
eIF4, eIF3, eIF4-9
eIF2
cap-binding factors
EF-Tu
EF-Ts
EF-G
eEF1
eEF1
eEF2
RF-1
RF-2
RF-3
eRF
Elongation
Termination
Diphtheria toxin
• a protein that catalyzes modification of eEF2, inhibiting eukaryotic protein synthesis
• Adds ADP ribose, blocking eEF2 ability to carry out translocation
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 3
Protein Synthesis in eukaryotes
1) Eukaryotic translation has many similarities to prokaryotic protein synthesis.
2) Notable differences include different ribosome size/composition, soluble protein factors,
and the mechanism of initiation.
3) Eukaryotic genes typically code for one 1 protein, while
many prokaryotic genes are polycistronic.
4) Eukaryotic transcription and translation are separated in space and time.
5) Most antibiotics are selective for bacterial protein synthesis.
Posttranslational processing of proteins
1.
2.
3.
4.
5.
Protein folding
Proteolytic cleavage
Amino acid modification
Attachment of carbohydrates
Addition of prosthetic groups
Control of gene expression: Take Home Message
1) Gene expression in all organisms is mainly controlled at the level of
transcription initiation.
2) Two 2 types of gene expression exist: constitutive and inducible/repressible genes.
3) Control of transcription rates is achieved by binding of the regulatory proteins to
promoter region, which turns RNA polymerase ‘on’ or ‘off’.
4) Regulatory proteins contain common structural motifs that enable them
to recognize specific regions of DNA via noncovalent interactions.
Flow of genetic information
DNA  RNA  Proteins  Cellular action
Replication 
transcription
 nucleare
translation
Reverse transcription
 
of telomeres
Notable exception: retroviruses
RNA  DNA  RNA  Proteins  Cellular action
Reverse transcription transcription
translation
 
nucleare
Regulation of gene expression in bacteria
 rate of protein synthesis in E. Coli can vary 1000-fold
 Gene activity is regulated on the level of transcription
Two 2 main types of prokaryotic genes:
1. 1. Constitutively expressed genes (housekeeping genes) – always on
R 2. Regulated genes – turned on in response to environmental conditions
Biochemistry of Medicinals I
Phar 6151
-galactosidase
H
H
H
O
H
H
H 6H
O H
1
4
O
OH H

O
H
H
H
H
H
H
H
O
 cis shape
O to C6 related
1 OH
OH H 
H
H
1
-lactose
because this
OH is 
OH
OH H 
H
H
O O6 H
4

O
H
-galactosidase  +

Page 4
of E. Coli is an example of inducible enzyme
H
O
H
H
controle Gene Expression
-1,4-glucoside
bond

H
O O6
H
4
Part 1
2O
H H
H O6
H
4
cis shape 
to C6 related
O
OH
O
H H
1 OH
H O
H
H
OH H 
( ) permease
==|=======|= = =|=====|=====|=========|===========|============|=========|= = DNA
*******
 ********** *********** ************
mRNA
∆∆∆∆∆∆

∆∆∆∆∆∆∆∆
∆∆∆∆∆∆∆∆∆ ∆∆∆∆∆∆∆∆∆∆
protein
repressor


 -gal'ase
lac-permease S-gal-trans-Ac
protein
LAC Lactose
 Splits LAC into Pumps LAC into Not involved in
removed

Gal + Glc
the cell
LAC metabolism
Promoter
Binding site for RNA Pol
that
when
(
)
Regulator repressor gene
,
,
Structural genes
prevents
repressor is bound
Codes for the repressor protein
Code for-galactosidase,
galactoside permease, and
thiogalactoside transacetylase,
respectively
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 5
Control of transcription in lac operon
IR
——
O
P
 mRNA 
IR
——
Z

O
P

H
A
IPTG
Z
1,6-allolactose =
H
Y
Y
A

IPTG
H
O O 6 H -1,4-glucoside bond
4
H
O 6H
H HO
 H
1
H
O
4
H
H
OH H O
H HO
1
O
H
H
H
H
-lactose
because this
is  OH
OH
OH H

H
H
O O6
H
4
H
H
O
H
H
H
 S
H HO
1
H
OH H
H
H
H
H
Prokaryotic mRNAs often are polycistronic
(encode more than one polypeptide)
(a) transcription in the presence of an
RNA polymerase binds, transcription of structural genees begins
Inactivated repressor cannot bind to
molecules bind to repressor, inactivating it Beta-Galactosidase Permease Acetyl transferase
Interactions between lac repressor protein and operator sequence
• Lac repressor protein is a tetramer of identical 37 kd subunits
• It tightly binds
sequence (Kd = 10-13 M)
• Finds
by diffusing along DNA molecule
• High selectivity for
(106)
5’ …GAA TTGTGACGGATAACAATTT…3’
3’… CTT AACACTGCC TATTGTTAAA…5’
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 6
LAC repressor 1LCC.PDB
C chaine 5'-CGCTCACAATT-3'
B chaine 3'-GCGAGTGTTAA-5'
Lac
repressor
crystal
structure
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Lac repressor bends DNA
Page 7
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 8
Zinc Finger
"INDEPENDENCE OF METAL BINDING BETWEEN TANDEM CYS2HIS2 ZINC FINGER DOMAINS"
B.A. KrizekL.E. Zawadzkeand J.M. Berg
Contents of file KRIZEK.KIN:
Kin.1 - Calpha trace of the Zif/268-DNA complex structure
Kinemage 1 is a Calpha trace of the three zinc finger
domains of Zif/268 bound to DNA, used for comparison
with the series of peptides containing two tandem zinc
finger domains. These peptides were examined to determine
the degree of metal binding cooperativity, if any, between
tandem domains. View1 corresponds to the orientation of
domains 2 and 3 in figure 1 of the manuscript, and includes
domain 1. The DNA can be turned on. The three metal
sites are displayed with their cysteine and histidine ligands.
One can highlight the relatively-conserved Arg27 (domain 1)
and Arg55 (domain 2), which participate in interfinger
hydrogen bonds with the backbone carbonyls of Ser45
(domain 2) and Ala73 (domain 3), respectively. An
additional interfinger contact, namely the residues which
make up the hydrophobic core between domain 2 (Thr52
and 58) and domain 3 (Pro62 and Phe63), can be turned on.
Coordinates from Brookhaven Data Bank file: 1ZAA
(Zif/268-DNA)
1D66.PDB-Zn2+-Gal4
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 9
Leucine zipper
Kine mage 3 shows the GCN4 leucin e
zippe r dimer (2ZTA), init iallyvie wed
from the side. The t wo ch ain s have
id enti cal sequ ence s and are o riented
parall el, with the N-termini at th e top.
The smoothbend ingand mu tual
coilingo f t he he lic es canbe
empha siz ed bytu rningo ff t he side
chain s, tu rningon the ax es, and
rotating the i mage. The supe rcoiling
allo ws the he lic es to s tayth e same
distance apart alongth eir entire
length; incont rast, mo st pa ir ed
he lic es inglobul ar prote ins are
straighte r and dive rge to ward th e
end s.
Turn the sidechains back onand notice that the interhelical contacts are made by sets of residues that look
like wide rungs on a ladder (notin factlike teeth on a zipper). Every other rung contains a pair of orange
leucines; alternate rungs contain pairs of gold valineswith one Met pair at the top and one Asn pair near the
middle. In this kinemageall of the Leu zipper sidechains are grouped and color-coded by their position in the
seven-residue heptad repeat: 'd' (Leu) in orange; 'a' (Val) in goldor in hotpink for Asn 16; 'e' and 'g'the contactedge hydrophilicsin skyblue; and the outside positions 'b''c'and 'f' in cyan. Turn on the "heptad lbl" button to see
labels a through g for one repeat.
An end view of the Leu zipper supercoil shows the helix-helix contacts most clearly. Choose Reset2 under
Graphics"zoometc" under the Other menuand zoom to enlarge the image. Turn off the "eg" and the "bcf"
buttonsto concentrate on the buried contact layers at positions a and d. For a detailed tourset the zoom to 3.0the
zslab to 100and move the slider to the top of the ztrans scrollbar; you should see just a little backbone and the
sidechains of Met 2 at the end of each helix. Then hold down the mouse just above the arrow at the bottom of
the ztrans scrollbar to move the structure fairly rapidly through the visible slab. A little more than one "rung"or
layerwill be in view at a time. Notice how similar the geometry is for each rung of Leu 'd' sidechains; Val 'a'
rungs are also similar to one anotherbut different in detail from Leu rungs. Leu Cbetas point toward each other
and their Cgammas turn out; Val Cbetas point outwardwhile one of their Cgammas points inward to touch. Val
is too short for optimal contact in a 'd' positionand Leu would not work in an 'a' positionbecause although its
Cgamma could lie in the correct place its Cdeltas would then bump into the opposite helix.
Turn on the "eg" side chains and move the structure through the slab again by holding down the mouse just
below the up arrow on the ztrans scrollbar. Notice how a given leucine contacts four surrounding sidechains on
the opposite helix: its symmetry-mate Leu in 'd'the two adjacent 'a' residuesand the preceding 'g' residue. This
kind of arrangement is called "knobs-into-holes" packing. It is usually not found in such a regular form in
globular proteinsbut was originally proposed by Francis Crick in 1953 to stabilize coiled coils.
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 10
-Sh eet
Helix -Turn-Helix
Helix -Loop-Helix
Catabolite repression
• The level of -galactosidase is low in the presence of glucose
• Glucose suppresses lac
transcription by lowering the
concentration of cyclic AMP (cAMP)
• The function of cAMP in bacteria is to activate CAP (Catabolite gene Activator Protein)
• cAMP-CAP complex binds promoter sitesstimulating the transcription of lac
H N H
N
N
H
H
O
O P
O
N
O
H
N
cAMP
H
O
O H
Promoter sequences for different genes
Promoter for:
trp
G
-35 region Spacer
TTGACA N17
tRNATyr
TTTACA
TTGACA
TTTACA
TTGATA
TTCCAA
TTGACA
TTGACA
P2
lac
rec A
lex A
T7A3
G
G
C
C
G
G
CONSENSUS
N16
N17
N17
N16
N17
N17
-10 region
TTAACT
TATGAT
GATACT
TATGTT
TATAAT
TATACT
TACGAT
TATAAT
Spacer
N7
N7
N6
N6
N7
N6
N7
 Transcribed
A
A
G
A
A
A
A
Biochemistry of Medicinals I
Phar 6151
Part 1
controle Gene Expression
Page 11
Enhancers can stimulate transcription from many nucleotides away
Enhancers
Looping of DNA
-200
|
-100
|
+1
|


Late RNA 
SV40 ——————————————
GC

DHFR —————
 Early RNA
———————————
box

 RNA
————
GC
—————
——————
—————
box


 RNA
Heat-shock gene — ——— —————
——
———

TATA
Heat-shock element
1. Steroid hormone response elements (HRE) Estrogenprogesteroneglucocortecoids
2. P53 tumor suppressor gene.
Control of gene expression: Take Home Message
1) Gene expression in all organisms is mainly controlled at the level of
transcription initiation.
2) Two 2 types of gene expression exist: constitutive and
/ repressible genes.
3) Control of transcription rates is achieved by binding of the regulatory proteins to
promoter regionwhich turns RNA polymerase ‘on’ or ‘off’.
4) Regulatory proteins contain common structural motifs that enable them to recognize
specific regions of DNA via noncovalent interactions.