Download Questions, chapter 14

Chapter 14 Answers
1.
Translation is much more complicated than transcription or DNA replication for
multiple reasons. For example, in contrast to transcription or replication, where the structural
complementarity of the bases means that the polymerase enzymes can simply let the template
guide the selection of nucleotides to be added to the RNA or DNA, the amino acids added
during translation have no structural relationship to the nucleotides that encode them. This
necessitates the presence of adaptor molecules—tRNAs—that can interact with both amino
acids and with mRNA to bridge the structural gap between the two. Also, the translation
machinery must select between 20 possible amino acids when synthesizing a new
polypeptide, in contrast to just four nucleotides during replication and transcription. This
means that a large array of tRNA molecules must be used in order to read the 61 different
amino acid-encoding codons and insert the correct amino acid.
2.
Eukaryotic mRNAs almost always contain a single open reading frame (i.e., they are
monocistronic), whereas prokaryotic mRNAs frequently include multiple open-reading
frames (i.e., they are polycistronic).
This difference is consistent with the different translation initiation mechanisms used
by the two types of organisms. For example, in prokaryotes, translation is initiated when the
small ribosomal subunit binds directly to a sequence just upstream of the start codon, called
the ribosome binding site. Because the ribosome can bind to this site even when it is located
at an internal position within an mRNA, the translation machinery can translate multiple
open-reading frames within a single message with equal efficiency. In eukaryotes, in contrast,
the ribosome first binds to the cap at the 5' end of the mRNA, and then scans along the
mRNA until it finds a 5'-AUG-3', which it then uses as a start codon. For this reason, the 5'
most AUG in an mRNA is generally the only codon at which translation can begin, meaning
that eukaryotic mRNAs can effectively only include a single open-reading frame.
The ability of prokaryotes to synthesize multicistronic mRNAs means that related sets
of proteins can be regulated in concert through the regulation of a single promoter. For
example, in E. coli the presence of the sugar lactose (and absence of glucose) leads to the
activation of a single promoter (the lac promoter) that drives the expression of a polycistronic
mRNA. This mRNA includes multiple genes that work together to metabolize the sugar. In
eukaryotes, this particular type of regulation is impossible, as individual promoters only
control the expression of single genes. Individual eukaryotic regulators can still modulate the
expression of multiple target genes, however, if each of the target genes' promoters contains a
binding site for the regulator. In this way, multiple copies of the regulator protein can
separately bind to the various promoters, together controlling the expression of all of the
target genes.
3.
The Shine-Dalgarno sequence, also called the ribosome binding site, is a sequence
element (ideally, 5'-GGAGG-3') that is found just upstream (usually, 3 to 9 nucleotides away)
of the start codon in many prokaryotic genes.
The sequence is recognized by the small ribosomal subunit (specifically, by a region
of the 16S RNA that contains the complementary sequence 5'-CCUCC-3'), allowing the
subunit to bind to the mRNA near the start codon with the proper positioning.
The Shine-Dalgarno sequence can vary considerably, both in terms of its sequence as
well as its distance from the start codon. Sequences that are most similar to the ideal
sequence—which is perfectly complementary to the 16S RNA sequence—tend to have the
most activity, while sequences that deviate from the ideal are generally less active. Also,
sequences that have the proper spacing relative to the start codon (i.e., 3 to 9 bases away) are
typically more active than those at other locations.
4.
The 5' cap acts to recruit the small subunit of the ribosome to the mRNA. Once bound
to the cap, the small subunit scans along the RNA until it encounters a start codon.
The cap also servers to protect mRNA from degradation in the cell, as RNAs lacking
a cap are rapidly destroyed by 5' to 3' exonucleases.
5.
tRNAs all share a secondary structure that resembles a cloverleaf, including a stem,
three stem-loops, and a variable loop. The stem of the cloverleaf, called the acceptor stem, is
made up of the 5' and 3' ends of the tRNA, and is the site of amino acid attachment to the
tRNA. The three stem-loops include the U loop, which usually contains a pseudouridine
base, the D loop, which contains dihydrouridine bases, and the anticodon loop, which
includes the three-nucleotide anticodon sequence that forms base pairs with codons in the
mRNA. The variable loop can be anywhere from 3 to 21 nucleotides in size. These domains
assemble to form an L shaped structure, in which the acceptor stem and the stem of the U
loop form one extended helix, and the anticodon stem and the stem of the D loop form a
second helix that extends away from the first helix at a 90 degree angle.
Several kinds of interactions help stabilize the tRNA structure, including base pairing
(including non-Watson-Crick pairing) within the helical regions, stacking interactions
between the bases of the base paired regions, and other interactions between the bases and the
sugar-phosphate backbone.
6.
Aminoacyl tRNA synthetases charge tRNAs in two steps. In the first step, called
adenylylation, the amino acid reacts with ATP in a way that adds an AMP molecule (through
its phosphate group) to the carbonyl group of the amino acid. This reaction also releases
pyrophosphate, which is then hydrolyzed to help drive the overall reaction forward. In the
second step, called tRNA charging, the adenylylated amino acid reacts with tRNA,
transferring the amino acid to the 3' end of the tRNA (to either the 2' or 3' hydroxyl group),
and releasing AMP in the process.
A given tRNA synthetase can only charge a single kind of amino acid, which
typically means that an organism has 20 different synthetase enzymes. Some organisms,
however, have fewer than 20 tRNA synthetases, although they still manage to charge tRNAs
with all 20 amino acids. They can do this by initially charging the tRNAs with fewer than 20
amino acids (using the available tRNA synthetases), and then using other enzymes to modify
some of the charged amino acids to re-create all 20 amino acids. For example, some bacteria
lack a synthetase for charging tRNAs with the amino acid glutamine, and charge tRNAGln in
the cell with glutamic acid instead. A second enzyme then modifies the glutamic acid
attached to the tRNAGln molecules (but not the glutamic acid linked to tRNAGlu) to convert
the glutamic acid to glutamine.
7.
The phenotype could be caused by any one of several possible types of mutations.
First, a mutant tRNA synthetase could be erroneously charging tRNAGlu with glutamine
(instead of glutamic acid), causing glutamine to be incorporated at glutamic acid-encoding
codons. If this were the case, the mutation could either be affecting the tRNA synthetase that
normally charges tRNAGlu with glutamic acid, causing it to sometimes select glutamine
instead, or it could be affecting the synthetase that normally charges tRNAGln with glutamine,
causing it to mistakenly select tRNAGlu. An additional possibility is that the cell contains an
enzyme that converts glutamic acid-tRNAGln to glutamine-tRNAGln (as discussed in the
previous question), and that the mutation lies within this enzyme, causing it to erroneously
convert glutamic acid substrates that are correctly attached to tRNAGlu.
You could try to experimentally address these three possibilities by cloning the genes
encoding the tRNA synthetases responsible for charging glutamic acid and glutamine, as well
as (if it exists) the enzyme that converts glutamic acid to glutamine on tRNAGln. Other than
simply examining the sequences of the genes to see if they contain any obvious mutations,
you could also examine the activities of the encoded proteins to see if they show any errors in
their substrate specificities. For example, you could incubate either of the tRNA synthetases
with tRNAGlu and with glutamine, and ask whether the tRNA ever gets charged. Also, if the
cell contains the enzyme that converts glutamic acid-tRNAGln to glutamine-tRNAGln, you
could ask whether the enzyme is ever capable of converting the glutamic acid present within
correctly charged tRNAGlu molecules.
8.
The ribosome moves in a 5' to 3' direction along the mRNA during translation.
The movement of the ribosome along the mRNA occurs during the translocation step
of translation. Just prior to translocation, the growing polypeptide is transferred from the
peptidyl-tRNA at the P site of the ribosome to the aminoacyl-tRNA at the A site. This causes
the tRNAs to shift their positions so that their 3' ends (the end attached to the amino acid or
polypeptide) now occupy the adjacent site in the large subunit, but with their anticodon ends
remaining in the original position (for example, for the peptidyl-tRNA, the 3' end occupies
the P site and the anticodon end occupies the A site). At this stage, the elongation factor EFG steps in, moving the anticodon end of the peptidyl tRNA so that both ends of the tRNA
occupy the P site. This forces the adjacent tRNA to move as well, so that it fully occupies the
E site. One consequence of the this movement is that the mRNA moves along with the
tRNAs, shifting its position relative to the ribosome by 3 base pairs in the 5' to 3' direction.
9.
The ribosome cycle begins when the small subunit of the ribosome binds to an
mRNA. The small subunit then recruits a large subunit to form a complete ribosome around
the mRNA. The ribosome then begins protein synthesis, moving from the start codon in the 5'
to 3' direction along the mRNA, adding one amino acid to the growing polypeptide chain for
each successive codon on the RNA. When it reaches a stop codon, the ribosome releases the
polypeptide chain, lets go of the mRNA, and dissociates to reproduce the large and small
subunits.
10.
The circularization of eukaryotic mRNAs is mediated by binding between eIF4F, a
three-subunit protein that interacts with the mRNA's 5' end, and poly-A binding protein,
which coats the 3' end. Accordingly, the mutation could be disrupting the interaction between
these two proteins, perhaps by altering the binding site of one of the two interacting partners.
The circularization helps increase the efficiency of translation, because it places
ribosomes terminating translation and dissociating from the mRNA in the proximity of the 5'
end of the same message. This increases the likelihood that the ribosome will re-bind the
message and begin another round of translation. A mutation that eliminates the circularization
of mRNAs, therefore, would eliminate this probability, requiring that a new ribosome find
and bind to the mRNA prior to each new round of translation. This would likely decrease the
efficiency of translation off of any given mRNA.
11.
The A site of the ribosome binds to the aminoacylated tRNA (i.e., the charged tRNA),
the P site binds the peptidyl-tRNA (i.e., the tRNA to which the polypeptide chain is linked),
and the E (for exit) site is occupied by the deacylated (i.e., uncharged) tRNA from which the
growing polypeptide chain has just been transferred. Each of the three sites is formed at the
interface between the large and the small ribosomal subunits.
The A, P, and E sites are occupied in the following ways at the indicated stages of
translation:
a.
A, E sites empty, P site containing fMet-tRNAifMet
b.
A site empty, P site containing peptidyl-tRNA, E site containing deacylated
tRNAifMet
c.
A site containing aminoacyl tRNA, P site containing peptidyl-tRNA, E site
containing deacylated tRNA;
d.
A site of large subunit empty, A site of small subunit containing anticodon
end of peptidyl-tRNA; P site of large subunit containing protein-linked end of peptidyltRNA, P site of small subunit containing anticodon end of deacylated-tRNA; E site of large
subunit containing acceptor stem end of deacylated tRNA, E site of small subunit containing
anticodon end of deacylated tRNA;
e.
A site containing puromycin, P site containing peptidyl-tRNA, E site
containing deacylated tRNA;
f.
A site containing release factor, P site containing peptidyl tRNA, E site
containing deacylated tRNA;
g.
12.
A site empty, P, E sites containing deacylated tRNA.
The ribosome contains three proofreading activities that help it ensure the accuracy of
amino acid incorporation. All of these act indirectly to ensure the accuracy of the
incorporated amino acid, acting specifically at the level of codon-anticodon pairing. The first
of these mechanisms involves interactions between two adjacent adenine residues of the 16S
RNA and the minor groove of the codon-anticodon complex. If (and only if) the base pairing
of the codon and anticodon is correct, these residues help stabilize the complex, increasing
the affinity of the tRNA for the ribosome. The second mechanism involves the GTPase
activity of EF-Tu. This activity, which is required for EF-Tu to release the bound tRNA and
leave the ribosome, is only stimulated when a correct codon-anticodon match is present. The
third mechanism functions once EF-Tu has left and the aminoacyl tRNA occupies the A site.
Here, the aminoacyl tRNA must rotate into the peptidyl transferase center of the ribosomal
large subunit in order for peptidyl transferase to occur. This rotation, however, frequently
causes tRNAs with incorrectly base paired anticodons to dissociate from the ribosome.
The accuracy of translation (with an error rate of 10-3 to 10-4) is somewhat lower than
that of transcription (10-4 to 10-5), and much lower than that of DNA replication (as low as 1010
). It makes sense that translation has the highest error rate because not only is translation the
most complex of the three processes, involving more potential sources of errors, but also
because mistakes that occur during protein synthesis only affect a single protein. Errors that
occur during transcription, on the other hand, are already more serious, because a single
mRNA can be used to synthesize multiple polypeptides. Finally, it makes sense that DNA
replication has the highest accuracy, because the permanent nature of the DNA in the nucleus
means that any error that arises during replication will be present in all of the cell's
descendants.
13.
Both EF-Tu and EF-G use the cycle of GTP binding and hydrolysis to regulate their
respective functions during translation. In its GTP-bound form, for example, EF-Tu
associates with aminoacyl tRNA and escorts it to the ribosome. When the aminoacyl-tRNA
enters the A site of the ribosome, and the correct codon-anticodon match is present, then the
ribosome activates the GTPase present within EF-Tu, causing the factor to hydrolyze the
bound GTP. In its GDP bound form, however, EF-Tu has much less affinity for aminoacyl
tRNA, and so it releases the tRNA and dissociates from the ribosome.
EF-G's properties also change depending on the form of guanine nucleotide that is has
bound. For example, EF-G only has affinity for the ribosome when bound to GTP, and binds
to the A site within the large subunit after the peptidyl transferase reaction. Once it binds to
the site, however, the ribosome stimulates the GTPase activity of the factor, provoking a
conformational change in the protein that allows it to contact the small ribosomal subunit and
shift the position of the tRNA. Following translocation, the altered ribosome has much less
affinity for EF-G (now in its GDP bound form), causing the factor to dissociate from the
ribosome.
14.
The stop codon is recognized in prokaryotic cells by the class I release factors RF1
and RF2 (UAG by RF1, UGA by RF2, and UAA by both RF1 and RF2). These factors are
composed entirely of protein, and use a particular stretch of three amino acids to form a
"peptide anticodon" that binds to and specifically recognizes the stop codon.
When the ribosome encounters a stop codon, RF1 or RF2 binds to the codon using its
three amino acid peptide anticodon, and another region of the protein (containing the short
amino acid sequence GGQ) interacts with the peptidyl transferase center of the ribosome to
effect the hydrolysis of the bond linking the polypeptide to the tRNA occupying the P site.
Once the polypeptide has been released, a class II release factor called RF3 binds to the
ribosome. RF3 is a GTP binding protein, and binds to the ribosome in its GDP-bound form.
The interaction with the ribosome causes RF3 to exchange GDP for GTP, with the result that
RF3 binds more strongly to the ribosome and displaces the class I release factor. With the
class I release factor gone, the ribosome stimulates the GTPase activity of RF3, causing it to
hydrolyze its bound GTP and then let go of the ribosome. Next, a protein called RRF binds
to the empty A site of the ribosome, where it recruits EF-G to help it displace the deacylated
tRNAs from the P and E sites. Once the tRNAs leave the ribosome, RRF, EF-G, and the
mRNA are all released by the ribosome. Finally, the initiation factor IF3 helps dissociate the
two ribosomal subunits from each other, thereby completing the translation cycle.
If the stop codon is missing, the ribosome translates to the end of the mRNA, passing
through the poly-A tail (and adding multiple lysines to the C terminus of the protein) and
stalling once it reaches the very end. A protein called Ski7 then dissociates the stalled
ribosome from the mRNA and recruits an exonuclease enzyme that degrades the defective
mRNA. The protein is then also destroyed, as proteins containing multiple lysines at their C
termini are targeted for rapid degradation in the cell.