Chap. 4. Basic Molecular Genetic Mechanisms (Part B)

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Chap. 4. Basic Molecular Genetic Mechanisms (Part
B)

• Topics
• Structure of Nucleic Acids
• Transcription of Protein-coding Genes and
  Formation of Functional mRNA
• Decoding of mRNA by tRNAs
• Stepwise Synthesis of Proteins on Ribosomes
• DNA Replication

Goals To learn the basic mechanisms of
transcription, RNA processing, translation, and
replication
Fig. 4.1.
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The Three Roles of RNA in Translation
Protein translation by ribosomes requires three
types of RNA (Fig. 4.17). Messenger RNA (mRNA)
specifies the amino acid sequence of the protein.
Each amino acid is selected based on the order of
triplet codons in mRNA. Transfer RNA (tRNA)
converts the information in mRNA codons into the
amino acid sequence of the protein. tRNAs carry
amino acids specified by the codons and base pair
with the codons via their anticodons. Ribosomal
RNA (rRNA) makes up the bulk of the mass of the
ribosome. One rRNA species (28S rRNA) is a
ribozyme that catalyzes the reaction in which the
peptide bond is formed.
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The Genetic Code
The codons for the 20 standard amino acids are
specified by triplets of bases known as the
genetic code (Table 4.1). Because there are 4364
possible combinations of triplet codons, most
amino acids are specified by more than one codon
(degeneracy). 61 codons specify amino acids.
Three do not (stop or termination codons).
Termination codons tell ribosomes where to end
translation of the mRNA. Most commonly, the AUG
codon (specifying methionine) serves as the start
codon, and tells the ribosome where to begin
translation. Few deviations from the standard
genetic code have been found, providing strong
evidence that life on earth evolved only once.
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Reading of the Triplet Code
There are three potential reading frames in all
mRNAs. However, only one reading frame is used
for translation, and is selected based on the
frame in which the AUG start codon appears.
Triplet codons are read in a non-overlapping,
comma-less manner (Fig. 4.18). Rarely are mRNAs
read in more than one frame. Likewise,
frame-shifting is very uncommon.
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Two-step Process for mRNA Decoding
Amino acids are attached in ester linkage to the
3'-terminus of tRNA, forming aminoacyl-tRNAs
(Fig. 4.19, step 1). The enzymes that carry out
this ATP-driven reaction are known as
aminoacyl-tRNA synthetases. Aminoacyl-tRNA
synthetases are highly accurate (high fidelity)
and this helps minimize translation errors. In
step 2, the amino acid is added to the growing
protein chain based on codonanticodon
interactions between mRNA and tRNA.
Bacteria synthesize 30-40 tRNAs, whereas
eukaryotes may synthesize 50-100. Thus, a given
amino acid often can be carried by more than one
species of tRNA. Each aminoacyl-tRNA synthetase
recognizes 1 amino acid and all of its cognate
tRNAs.
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Structure of tRNAs
tRNAs typically are 70-80 nucleotides in length.
They all have a cloverleaf secondary structure
and fold into an L-shaped tertiary structure
(Fig. 4.20). Four double-helical stems occur, and
three of these have loops of 7-8 residues at
their ends. One loop (the anticodon loop)
contains the anticodon. The upper stem is known
as the acceptor stem and ends with a CCA sequence
in all tRNAs. The amino acid is attached in ester
linkage to the 2' or 3' hydroxyl group of the A
residue. Many residues are modified in tRNA, and
some modifications are shown in the figure.
aa
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Codon-anticodon Base Pairing
H-bonding between the 1st and 2nd positions of
the codon and the 3rd and 2nd positions of the
anticodon nearly always occurs via Watson-Crick
base pairing. However, base pairing between the
3rd position of the codon and 1st position of the
anticodon (termed the "wobble position" in both
sequences) is less constrained (Fig. 4.21). For
example, G, U, and I (inosine) in the wobble
position of the anticodon can base pair with C/U,
A/G, and C/A/U in the codon, respectively. Wobble
base pairing reduces the number of tRNA genes
that an organism must make to carry out
translation. It also helps protect against
mutations that might inactivate tRNA genes.
Wobble is allowed at the codonanticodon
interaction site due to stabilization of
tRNA-mRNA binding by ribosomes.
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Ribosome Composition
Ribosomes are RNA-protein supramolecular
complexes. They are the most abundant type of
RNA-protein complex in cells. The compositions of
prokaryotic and eukaryotic ribosomes are
summarized in Fig. 4.22. Although proteins
outnumber rRNAs, rRNAs comprise 60 of the
ribosomal mass (see Fig. 4.23).
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Overview of Eukaryotic Translation Initiation
Like transcription, translation is
mechanistically divided into initiation,
elongation, and termination stages. All stages
require translation factors in addition to
ribosomes, mRNA, and aa-tRNAs. Prior to
initiation of translation, the 60S and 40S
subunits of the 80S eukaryotic ribosome occur in
their dissociated states. As described next, the
assembly of the 80S ribosome initiation complex
at the start codon of the mRNA proceeds via
binding of the mRNA and a charged Met-tRNAiMet
initiator tRNA to the 40S subunit, with
subsequent addition of the 60S subunit.
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Translation Initiation in Eukaryotes I
Translation initiation in eukaryotes begins with
three components/complexes that are shown near
the top of Fig. 4.24. These are 1) the 40S
ribosomal subunit, to which the eIF1, eIF1A, and
eIF3 initiation factors are bound 2) the
eIF2.GTP Met-tRNAiMet ternary complex and 3) a
circular mRNA formed by the binding of the eIF4
cap-binding complex at the 5 end of the mRNA to
poly(A) binding protein (PABP) associated with
the 3 end of the mRNA. These components
associate in Steps 2 and 4 of the diagram,
placing Met-tRNAiMet in the P site of the 40S
subunit.
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Translation Initiation in Eukaryotes II
In the next stage of initiation, the mRNA is
scanned in the 5 to 3 direction until the first
AUG start codon is brought into the P site (Steps
5 6). Then the hydrolysis of GTP by eIF2
generates a stable 48S initiation complex in
which the initiator tRNA (Met-tRNAiMet) is
H-bonded to the AUG codon.
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Translation Initiation in Eukaryotes III
In the final stages of initiation, all initiation
factors except eIF1A dissociate from the 48S
initiation complex and the 80S subunit and
eIF5B.GTP complex add on (Step 7). After eIF5B
hydrolyzes GTP, the last initiation factors
depart, and the stable 80S initiation complex is
created (Step 8). This complex contains the
complete E (exit), P (peptidyl-tRNA), and A
(aminoacyl-tRNA) binding sites, with Met-tRNAiMet
bound to the P site.
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Translation Elongation in Eukaryotes
Translation elongation requires the assistance of
elongation factors (Fig. 4.25). In Step 1 of
elongation, the second amino acid of the
polypeptide is carried to the A site of the
ribosome by an EF1a.GTP complex. It binds to the
mRNA via the anticodon located in the A site. In
Step 2, GTP is hydrolyzed and EF1a departs. In
Step 3, the 28S rRNA of the 60S subunit catalyzes
peptide bond formation (see Fig. 4.17), resulting
in a dipeptidyl-tRNA residing in the A site. In
Step 4, the factor EF2.GTP binds, the ribosome
translocates one codon along the mRNA, and GTP is
hydrolyzed. As a result, the dipeptidyl-tRNA is
placed in the P site, and the uncharged tRNAiMet
enters the E site. The uncharged tRNA is ejected
from the ribosome in the next cycle of elongation.
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Translation Termination in Eukaryotes
When a stop codon (UAA, UAG, UGA) enters the A
site, it is recognized and bound by the eRF1
release factor (Fig. 4.27). eRF1 forms a complex
with eRF3.GTP. Hydrolysis of GTP by eRF3 results
in cleavage of the linkage between the
polypeptide and peptidyl-tRNA and release of the
protein from the ribosomal post-termination
complex. A protein called ABCE1 then binds to the
complex, and via ABCE1 hydrolysis of ATP, the 40S
and 60S subunits are separated. The 40S subunit
recombines with the eIF1, eIF1A, and eIF3 factors
making it ready for another round of initiation.
Folding of the released polypeptide chain is
aided by chaperones (not shown).
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Polysomes Ribosome Recycling
Polypeptide chain elongation proceeds at a rate
of 3-5 amino acids per second. The efficiency of
translation is increased via the binding of
multiple ribosomes (polysomes) to the mRNA at a
given time (Fig. 4.28b). Translation efficiency
is further increased due to the complex between
poly(A)-binding protein (PABP) and the eIF4-mRNA
5'-cap that occurs in mRNA (Fig. 4.28b). This
circular complex positions ribosomes that have
just terminated translation of the message near
its 5' end. These ribosomes are recycled and
rapidly reinitiate another round of translation.
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Mechanism of DNA Replication
DNA is replicated via a semiconservative
mechanism (Fig. 4.29a). In this method the
parental DNA duplex separates, and each strand
serves as a template for synthesis of a
complementary strand. Thus the daughter DNA
molecules consist of one old one new DNA
strand. The alternative conservative model for
replication was ruled out based on a classic
experiment conducted by Meselson Stahl (Fig.
4.29b).
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DNA Synthesis at the Replication Fork
An overview of semiconservative replication is
presented in Fig. 4.30. The event depicted is
occurring at a replication fork formed after
replication has initiated at a replication
origin. One strand of the lower daughter molecule
(the leading strand) is being synthesized
continuously in the same direction as fork
movement. One strand of the upper daughter
molecule (the lagging strand) is being
synthesized in the opposite direction in a
discontinuous manner in relatively short segments
called Okazaki fragments.
DNA polymerases require primers for DNA
synthesis. Only one primer is needed for
synthesis of the leading strand. However, each
Okazaki fragment on the lagging strand is made
from a primer. Primers used in DNA synthesis are
composed of RNA DNA. Eventually, RNA primers
are replaced with DNA and Okazaki fragments
joined together by DNA ligase.
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Replication of SV40 Viral DNA (Part A)
The mechanism of eukaryotic replication is known
mostly from the study of the replication of the
SV40 virus, which infects monkeys (Fig. 4.31).
SV40 is a good model system because all but one of
the proteins required for its replication (viral
large T-antigen) are synthesized by host cells.
At SV40 replication forks, large T-antigen uses
its helicase activity to unwind DNA. Both strands
of single-stranded DNA are bound and coated by
replication protein A (RPA) which keeps the DNA
in a ideal template conformation (Fig. 4.31c).
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Replication of SV40 Viral DNA (Part B)
The leading strand is synthesized continuously by
DNA polymerase d (Pol d) (Fig. 4.31). Pol d forms
a complex with replication factor C (Rfc) and
proliferating cell nuclear antigen (PCNA) which
keep the enzyme bound to DNA (Fig. 4.31b). RPA is
displaced as the polymerase moves forward
synthesizing the chain in a 5' to 3' direction.
The lagging strand is synthesized discontinuously
in a
5' to 3' direction from RNA/DNA primers made by a
complex containing primase and Pol a. The 3' ends
of primers are elongated by a second Pol
d/Rfc/PCNA complex. RNase H degrades the RNA
primers, and the gaps are filled in by Pol d.
Nicks in the lagging strand are sealed by DNA
ligase. Topoisomerase I reduces positive
supercoiling ahead of large T-antigen.
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Bidirectional Replication of SV40 DNA
Replication of SV40, and most likely all other
prokaryotic and eukaryotic DNAs, occurs
bidirectionally starting from a replication
origin. Bidirectional replication increases the
rate at which DNA molecules are copied. The
bidirectionality of replication has been
demonstrated in experiments such as shown in Fig.
4.32. When a mixture of replicating SV40 DNA
molecules are linearized by cutting with a
restriction enzyme, the replication bubbles
observed all are centered at the same position on
the DNA. This indicates replication has proceeded
in both directions from the origin.
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Model for Bidirectional DNA Replication
A conceptual model for initiation of
bidirectional replication and fork movement away
from a replication origin is shown in Fig. 4.33.
For SV40, large T-antigen unwinds the parental
strands. In eukaryotic chromosomal replication,
cellular helicases known as MCM proteins perform
unwinding. Each eukaryotic chromosome contains
multiple replication origins separated by tens to
hundreds of kilobases. The activation of MCM
helicases (and thereby, DNA replication) is
controlled by S-phase cyclin-dependent kinases
(Chap. 19).