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A. The flow of genetic information in a cell is almost exclusively in one direction: DNA
S
RNA S protein.
B. The flow of genetic information follows a colinearity principle in that a linear sequence
of nucleotides in DNA is decoded to give a linear sequence of nucleotides in RNA which
is decoded to give a linear sequence of amino acids in a protein.
C. This flow involves three main successive steps called transcription, processing the
RNA transcript, and translation.
A. TRANSCRIPTION IN GENERAL
1.
Transcription is the mechanism by which the cell copies DNA into RNA and occurs
in the nucleus.
2.
During transcription, the double helix DNA is unwound and the DNA template
strand forms transient RNA–DNA hybrid with the growing RNA transcript. The
other DNA strand is called the DNA nontemplate strand.
3.
DNA sequences which flank the gene sequence at the 5
end of the template strand
are called upstream sequences. DNA sequences which flank the gene sequence at
the 3
end of the template strand are called downstream sequences.
4.
Transcription is carried out by DNA-directed RNA polymerase that copies a DNA
template strand in the 3
S 5 direction which in turn produces an RNA tran-
script in the 5
S 3 direction
.
5.
RNA polymerase differs from DNA polymerase in that RNA polymerase does not
need a primer and does not have 3
S 5 proofreading exonuclease activity.
6.
There are three RNA polymerases as follows:
a.
RNA polymerase I produces 45S rRNA.
b.
RNA polymerase II produces an RNA transcript that is further processed
into mRNA used in protein synthesis.
c.
RNA polymerase III produces 5S rRNA, tRNA, some snRNAs, snoRNA, and
miRNA (microRNA)
.
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34
CHAPTER 6
B. TRANSCRIPTION IN PROTEIN SYNTHESIS
(Figure 6-1).
1.
In protein synthesis, RNA polymerase II
produces an RNA transcript by a complex
process that involves a number of general
transcription factors called TFIIs (tran-
scription factors for RNA polymerase II).
2.
TFIID
binds to the TATA box which then
allows the adjacent binding of TFIIB.
3.
The next step involves TFIIA, TFIIE,
TFIIF, and TFIIH, and RNA polymerase
II engaged to the promoter forming a tran-
scription initiation (TI) complex.
4.
The TI complex must gain access to the
DNA template strand at the transcription
start site which is accomplished by TFIIH
(a DNA helicase).
5.
TFIIH also contains a protein kinase that phosphorylates (P) RNA polymerase II
so that RNA polymerase II is released from the TI complex.
6.
The TI complex will produce only a basal level of transcription or constitutive
expression. Other factors called cis-acting DNA sequences and trans-acting pro-
teins are necessary for increased transcription levels.
Processing the RNA Transcript into mRNA.
A cell involved in protein synthesis
will use RNA polymerase II to transcribe a protein-coding gene into an RNA transcript that
must be further processed into mRNA. This processing involves
A. RNA CAPPING is the addition of a 7-methylguanosine to the first nucleotide at the 5
end of the RNA transcript. RNA capping functions to protect the RNA transcript from
exonuclease attack, to facilitate transport from the nucleus to the cytoplasm, to facili-
tate RNA splicing, and to attach the mRNA to the 40S subunit of the ribosome.
B. RNA POLYADENYLATION is the addition of a poly-A tail (about 200 repeated AMPs)
to the 3
end of the RNA transcript. The AAUAAA sequence is a polyadenylation sig-
nal sequence which signals the 3
cleavage of the RNA transcript. After 3 cleavage,
polyadenylation occurs. RNA polyadenylation functions to protect against degradation,
to facilitate transport from the nucleus to the cytoplasm, and to enhance recognition
of the mRNA by the ribosomes.
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35
PROTEIN SYNTHESIS
C. RNA SPLICING
1.
RNA splicing is a process whereby all introns (noncoding regions; intervening
sequences) are removed from the RNA transcript and all exons (coding regions;
expression sequences) are joined together within the RNA transcript.
2.
RNA splicing requires that the intron/exon boundaries (or splice junctions) be rec-
ognized. In most cases, introns start with a GT sequence and end with an AG
sequence (called the GT–AG rule).
3.
RNA splicing is carried out by a large RNA–protein complex called the spliceo-
some which consists of five types of snRNAs (small nuclear RNA) and
50
different proteins. Each snRNA is complexed to specific proteins to form small
nuclear ribonucleoprotein particles (snRNPs).
4.
The RNA portion of the snRNPs hybridizes to a nucleotide sequence that marks
the intron site (GT–AG rule), whereas the protein portion cuts out the intron and
rejoins the RNA transcript. This produces mRNA that can leave the nucleus and
be translated in the cytoplasm.
5.
There are two type of spliceosome: the major GU–AG spliceosome which splices
GT–AG introns and the minor AU–AC spliceosome which splices the rare class of
AT–AC introns.
is the mechanism by which only the centrally located
nucleotide sequence of mRNA is translated into the amino acid sequence of a protein and
occurs in the cytoplasm. The end or flanking sequences of the mRNA (called the 5
and 3
untranslated regions, 5
UTR and 3UTR) are not translated. Translation decodes a set of
three nucleotides (called a codon) into one amino acid (e.g., GCA codes for alanine, UAC
codes for tyrosine). The code is said to be redundant which means that more than one
codon specifies a particular amino acid (e.g., GCA, GCC, GCG, and GCU all specify alanine
and UAC and UAU both specify tyrosine).
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36
CHAPTER 6
● Figure 6-2 Translation. This diagram joins the process of translation at a point where three amino acids have already
been linked together (amino acids 1, 2, and 3). The process of translation is basically a three-step process that is repeated
over and over during the synthesis of a protein. The enzyme aminoacyl-tRNA synthetase links a specific amino acid with
its specific tRNA. In step 1, the tRNA and amino acid complex 4 binds to the A site on the ribosome. Note that the
direction of movement of the ribosome along the mRNA is in a 5
S
3
direction. In step 2, the enzyme peptidyl trans-
ferase forms a peptide bond between amino acid 3 and amino acid 4 and the small subunit of the ribosome reconfig-
ures so that the A site is vacant. In step 3, the used tRNA 3 is ejected and the ribosome is ready for tRNA and amino
acid complex 5.
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37
PROTEIN SYNTHESIS
A. Translation uses transfer RNA (tRNA; Figure
6-3). tRNA is a cloverleaf structure consisting of
about 75–90 nucleotides and has four arms. The
acceptor arm contains the amino acid-binding
site, whereby the amino acid covalently binds at
the CCA 3
end. The anticodon arm contains
the anticodon trinucleotide in the center of the
loop that base pairs with the codon on mRNA.
The anticodon arm demonstrates tRNA wobble,
whereby the normal A-U and G-C pairing is re-
quired only in the first two base positions of the
codon, but variability or wobble occurs at the
third position. The T
C arm is defined by this
trinucleotide of thymine (T),
(pseudouridine;
5-ribosyl uracil), and cytosine (C). tRNA is the
only RNA where thymine is present. The D arm
is named because it contains 5,6-dihydrouridine
(D) residues.
pseudouridine (5-ribosyl
uracil) m
5
C
5-methylcytidine m
1
A
1-
methyladenosine D
5,6-dihydrouridine.
● Figure 6-3 Transfer RNA (tRNA).
A
OH 3’ end
C
C
A
C
Acceptor arm
amino-acid-binding
site
5’ end
G
U
A
A
C
C
P
G
C
A
U
m
m1
m5
m5
m5
m5
T C arm
U
G
G
G
C
C
A
G
G
G
U
G
C
G
G
G
C
C
C
A
U
C
C
U
U
G
G
U
A
U
A
U
D-arm
Anticodon arm
Anticodon
G
C
A
A
D
G
U
G
G
G
G
G
G
C
C
G
C
G
C
U U
A
G
C
T
G
B. Translation uses the enzyme aminoacyl-tRNA synthetase which links an amino acid
to tRNA. tRNA charging refers to the fact that the amino acid–tRNA bond contains the
energy for the formation of the peptide bond between amino acids. There is a specific
aminoacyl-tRNA synthetase for each amino acid. Because there are 20 different amino
acids, there are 20 different aminoacyl-tRNA synthetase enzymes.
C. Translation uses the enzyme peptidyl transferase which participates in forming the
peptide bond between amino acids of the growing protein.
D. Translation requires the use of ribosomes which are large RNA–protein complexes that
consist of a 40S subunit (consisting of 18S rRNA and
30 ribosomal proteins) and a
60S subunit (consisting of 5S rRNA, 5.8 rRNA, 28S rRNA, and
50 ribosomal pro-
teins). The ribosome moves along the mRNA in a 5
S 3 direction such that the NH2-
terminal end of a protein is synthesized first and the COOH-terminal end of a protein
is synthesized last.
E.
Translation begins with the start codon AUG that codes for methionine (the optimal
initiation codon recognition sequence is GCACCAUGG) so that all newly synthesized
proteins have methionine as their first (or NH2 terminal) amino acid which is usually
removed later by a protease.
F.
Translation terminates at the stop codon (UAA, UAG, UGA). The stop codon binds re-
lease factors that cause the protein to be released from the ribosome into the cytoplasm.
A. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
1.
SLE is a chronic autoimmune disease that affects the blood, joints, skin, and kid-
neys. SLE occurs predominately in women of childbearing age.
2.
SLE pathogenesis involves polyclonal B cell activation, sustained estrogen activity,
and environmental factors such as the sun or the drug procainamide (drug-
induced SLE).
3.
Laboratory findings include
a.
Anti-double-stranded DNA antibodies.
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