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Maivel H. Ghattas, MD, MSc Pediatrics Prof. Medical Biochemistry, Faculty of Medicine, PSU

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DNA
Ribosomal RNA gene
RNA polymerase I
Pre-rRNA
(28S)
(5.8S) (18S)
Cleavage by
RNases
28S
5.8S 18S
Ribosomal RNAs
Figure 30.15
Posttranscriptional processing Of eukaryotic ribosomal RNA by ribonucleases.

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A 16-nucleotide
sequence at the
5'-end (shown in
green) is cleaved
by RNase p.
A 14-nucleotide
intron (shown in
purple) in the
anticodon loop
is removed by
splicing.
Figure 30.16
Uracil at the
3' end are replaced
by the CCA sequence
found in all mature
tRNAS.
Many bases
are converted
to characteristic
bases
(shown in
yellow).
Pre-tRNA
Mature tRNA
A. Primary tRNA transcript. B. Functional tRNA after posttranscriptional modification. Modified bases include D
(dihydrouridine), (pseudouridine), and m, which means that the base has been methylated.

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O
H2N N
5'-End
9-13
o
Triphosphate linkage
0-+-O-Å-O-Å-O
SCH2
Base
Coding region
for protein
Polyadenylation
signal sequence
OH OH
p-0
AAUAAAAAAAAA••••••• A OH
7-Methylguanosine triphosphate "cap"
Figure 30.17
mRNA
Poly-A tail
Posttranscriptional modification of mRNA showing the 7-methylguanosine cap and poly-A tail.

The poly-A tail is inserted by polyadenylate polymerase

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snRNP.
snRNPs
ATP
z-OH A at
the spuee
dm site of
snRNPs
o' 2 at tho
m RNA
Figure 30.18
Removal of introns. snRNP
small nuclear ribonucleoprotein
particle.

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snRNA combine with proteins to form snRNP snRNPs facilitate splicing

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TRANSCRIPTION
rRNA
TRANSLATION
Figure 31.1
Protein synthesis or "translation."

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A codon is a three letter word that codes for an amino acid. Met Ser Pro ***

There are 4 nucleotides, and 3 nucleotides per codon, so there are 4 3  = 64 combinations.

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MIDDLE BASE
5' - BASE
four
rows show
sixteen amino
acids whose
codons
with A.
This column
shows sixteen
amino acids
whose codons
have the middle
base U.
Figure 31.2
3' - BASE
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Me
Val
val
val
val
Ser
Ser
Ser
Ser
pro
Pro
pro
pr0
Thr
Thr
Thr
Thr
Tyr
Tyr
Stop
Stop
His
His
Gin
Gin
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
cys
Stop
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
These four,
separated
rows show
amino
acids whose
codons end
with G.
The codon 5-AUG-3 designates
methionine (Met).
Use of the genetic code table to translate the codon AUG.

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Specific : a specific codon always encodes the same amino acid Universal : The code is the same for all organisms Redundant : A given amino acid may be encoded by more than one codon Nonoverlapping : Read from a starting point as a continuous sequence, taken three at a time ABCDEFGHIJKLMNOPQRSTUVWX ABC/DEF/GHI/JKL/MNO/PQR/STU/VWX

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ULJA
(Termination codon)
Nonsense
mutation
(Codon for serine)
Missense
mutation
Silent
mutation
(Codon for serine)
(Codon for proline)
31 a
ot a in ttw ot
mRMA

Silent Mutation : encodes same amino acid Missense Mutation : encodes a different amino acid Nonsense Mutation : encodes a stop codon

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An insertion or deletion of one or two bases AUG  UCA  CCA UUU UAC Met Ser Pro Phe Tyr AUG  UC C  A CC AUU UUA C Met Ser Thr Ile Leu

Addition of base
mRNA
UCA
C CU AUG GCUATØ
ser
ser Tyr Gly
Addition Of U
UCACCUAUGGCU
Ser
Pro Met Ala
5'-End
3'-End
Deletion of C
c
UCACUAUGGCU
Ser Leu Trp
Deletion of base
Figure 31.5
Frame-shift mutations as a result Of a"tion or deletion Of a base can
an alteration in the reading frame Ot mRNA.

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NONPOLAR SIDE CHAINS
•H3N-c-C00H
9.6 pK, -23
G *cine
•HB- C -COOH
•H3NA3 -COOH
Tryptophan
Figure 1.2
-ccx)H
Isoleucine
•HSN-C
CH2
CH2
Methionine
•HaN-c-C00H
Hac
Valine
*HB-c-COOH
•H2N
H2C
CH2
Proline
of the twenty amino acids found in proteins according to the charge and
gxylarity of their side chains is shown here and continues in Figure 1.3. Each amino acid
is shown in its fully protonated form with disscxiable hydrogen ions represented in
print. The PK values for the a-carmxyl and a-amino groups of the nonpolar amino acids
are similar to those shown for glycine.

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Amino Acids tRNA At least one specific type per amino acid At least 50 in humans 30-40 in bacteria

Methionine
ACC
3'-End
Anticodon
(5'-CAU-3')
5'-End
UAC
5'-End
Complementary
(antiparallel)
binding
mRNA
3'-End
Codon
Figure 31.6
Complementary. antiparallel binding of the anticodon for
methionyl-tRNA (CAU) to the mRNA for methionine (AUG).

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The codon is 5’-AUG-3’.  What is the anti-codon sequence of the tRNA that would recognize this codon? 5’-AUG-3’ 5’-UAC-3’ 5’-CAU-3’ 5’-TAC-3’ 5’-CAT-3’

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Methionine
ACC
3'-End
Anticodon
(5'-CAU-3')
5'-End
UAC
5'-End
Complementary
(antiparallel)
binding
mRNA
3'-End
Codon
Figure 31.6
Complementary. antiparallel binding of the anticodon for
methionyl-tRNA (CAU) to the mRNA codon for methionine (AUG).

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Amino acid
ATP
Aminoacyl-tRNA
(E)
p Pi 2Pi
E-AMP—Amino acid
AMP
CA—Amino acid
Aminoacyl-tRNA
Figure 31.7
Attachment Of a specific amino acid to its
tRNA by aminoacyl-tRNA

Extremely High Fidelity Reads both the three letter genetic code and the 20 letter code of the amino acids Has “proofreading” capability in that it can remove a mischarged amino acid

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Amino Acids tRNA mRNA Ribosomes Protein factors Initiation factors Elongation factors Termination factors

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Amino Acids tRNA mRNA Ribosomes Protein factors ATP and GTP 4 high energy bonds are required to add one amino acid

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5'-End
A CCUC
5'-End G G A u G
Shine-Dalgamo sequence
nS-Ribosomal subunit
Figure 31.10
16S-Ribosomal RNA
RNA
593'-End
Complementary binding betwæn prokaryotic mRNA Shine-Dalgarno sequence and 16S rRNA.

In eukaryotes, the 40S ribosomal subunit binds to the cap structure at the 5’ end of the mRNA and moves down the mRNA until the initiator AUG codon is encountered.

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CH3
A-C-C-NH2
Methionine
Initiator tRNA
N 10-Formyl-THF Transformyjase
THF
CCA
fMet-tRNA
Figure 31.11
Generation of the initiator
tRNA (fMet-tRNA).
CH3
CH2
CH2 H
N-Formyl-
methionine
o

In prokaryotes and mitochondria of eurkaryotes, the initiator tRNA carries a methionine that has been N-formylated IF-2 in  Escherichia coli In eukaryotes, the initiator tRNA carries methionine, but it is NOT N-formylated Several eIFs are involved in humans The initiator tRNA enters the ribosome at the P site. All other tRNAs enter at the A site.

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The protein is synthesized in the amino-terminal (NH 2 -) to the carboxyl-terminal (COOH) direction. The mRNA serves as a template and is read in the 5’ to 3’ direction.

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Pot"
Figure 31.12
Formation of a t»nd.

Because the 23S rRNA found in the 50S subunit of the ribosome catalyzes the peptidyl transferase activity, it is referred to as a ribozyme.

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Bind, to the 30S
its int&lering with
u G U UU AAGCGG
proten
IF-a
IF-I
IF-2
aid
Ot the
TETRACYCLNES'
"ith
tho to
INITIATION
GTP
tt-w
the 70S initiatia'
IF-2 IF-3
GTP GDP
ERNA to in
empty A.
Phenylal anyt•tRNA
Gop
EF-Tu
EF.Ts
ELONGATION
AUG
the
ch•n) mate
AAA
B a to
t RNA b. into
ct.in. æing ot
in both md
Ptw
A c AAA
CHLOR AM
n bits ßp
protein synth
Figure 31.13

The initiator tRNA aligns with the start codon at the P site of the ribosome.

Elongation factors direct the correct tRNA into the A site.

Peptidyl transferase catalyzes the formation of the peptide bond, moving the peptide chain from the P site to the A site.

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tMet
A u G U UU AAG
CLINDAMYCIN*
ERYTHROMYCINS
Bind to a
on the 50S
bacterial
inhibiting
along mRNA
DIPHTHERIA TOXIN
Inactivates the
eEF•2.
TERMINATION
RF.I
RF.2
RF.a
p peptide
acc
Steps 3, 4,
until
growing is
a
A termination codon
of the newty

The ribosome moves 3 bases in the 3’ direction on the mRNA, placing the peptidyl-tRNA in the P site, and the uncharged tRNA in the E site.

These steps repeat until a termination codon is reached.

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Streptomycin Binds to 30S subunit; interferes with initiation Tetracycline Blocks ribosome to aminoacyl-tRNA Puromycin Aminoacyl-tRNA homologue (eukaryotic, too) Chloramphenicol Inhibits peptidyltransferase Clindamycin and Erythromycin Binds to 50S subunit; inhibits translocation

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Diphtheria toxin Corynebacterium diphtheriae  must be infected with a bacteriophage ( b  corynephage) that carries the  tox  gene. Low grade fever, malaise, mild sore throat, grayish vegetative tonsillar growth. ADP-ribosylation of eEF-2

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Pseudomonas exotoxin A Pseudomonas aeruginosa Nosocomial infections in hosts with altered immune defenses Broad tissue specificity (burns, wounds, cornea, urinary tract, lungs) ADP-ribosylation of eEF-2

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Ricin Castor beans Removes adenine from 28S rRNA (eukaryotic) Potential use for bio-terrorism or biological warfare

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Ribosome encounters a stop codon in the A site In prokaryotes: RF-1 recognizes UAA and UAG RF-2 recognizes UGA and UAA RF-3 binds GTP and stimulates RF-1 and RF-2 In eukaryotes: eRF does it all

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Growing chains
NH2
NH2
mRNA
NH2
5-End
Figure 31.14
NH2
Ribosomß
NH2
COOH
NH2
3-End
A polyribosome consists of several ribosomes simultaneously translating one mRNA.

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7Me Gppp AAAAAAAAAAA 7Me Gppp AAAAAAAAAAA

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Phosphoryl anon
Glycosylation
0042-
Figure 31.15 (continued on next page)
Posttranslation modifications of some
amino acid residues.

Carboxylation
Biotin-linked enzyme
N •gH—C
Famesylated protein
Figure 31.15 (continued)
Posttranslation modifications of some
amino acid residues.

Trimming (Zymogens) Phosphorylation (Serine, Threonine, Tyrosine) Glycosylation O-linked (Serine, Threonine) N-linked (Asparagine) Hydroxylation (Proline, Lysine) Other (Carboxyl, Farnesyl)

Protein Synthesis