Chapter 27 Exam 2 Flashcards
What is Crick’s adaptor hypothesis?
adaptor hypothesis = postulation that a small nucleic acid could act as an adaptor, binding to both a specific amino acid and the mRNA sequence encoding that amino acid
verified with the discovery of tRNA
What is translation? (Translation and Aminoacyl-tRNAs)
translation = the overall process of mRNA-guided protein synthesis
the tRNA adaptor “translates” the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide
amino acids are “activated” for protein synthesis.
What are aminoacyl-tRNAs?
= tRNA attached to an amino acid
What are aminoacyl-tRNA synthetases?
catalyze the formation of aminoacyl-tRNAs
What is necessary to encode each amino acid?
Three Nucleotides are Necessary to Encode Each Amino Acid
the four code letters of DNA (A, T, G, and C) in groups of two yields only 42 = 16 different combinations
insufficient to encode 20 amino acids.
groups of three yield 43 = 64 different combinations
What is a codon?
codon = a triplet of nucleotides that codes for a specific amino acid
in all living systems, translation occurs in such a way that codons are read in a successive, nonoverlapping fashion
What is a triplet?
The Triplet, Nonoverlapping Code
inserting or deleting one base pair alters the amino acid sequence coded by the mRNA
inserting or deleting three nucleotides leaves the remaining triplets intact
provides evidence that a codon has only three nucleotides
What is reading frame?
reading frame = method of dividing nucleotides such that a new codon begins every three nucleotide residues
established by the first codon
no punctuation between codons
in principle, any given ssDNA or mRNA sequence has three possible reading frames
What was the first breakthrough of amino acids connected to codons?
Nirenberg and Matthaei fed poly(U) and 20 radiolabeled amino acids to E.coli
found that radioactive polypeptide only formed in the tube containing *Phe
thus UUU codes for Phe
the same approach revealed:
CCC codes for proline
AAA codes for lysine
GGG did not generate any polypeptide, because it forms tetraplexes that cannot be bound by ribosomes
What is polynucleotide phosphorylase?
polynucleotide phosphorylase = catalyzes the formation of RNA polymers with a base composition that reflects the relative concentrations of precursors in the medium
discovered by Ochoa
Nirenberg and Ochoa groups used artificial mRNAs made by polynucleotide phosphorylase to identify the base compositions of coding triplets
What can trinucleotides induce?
Trinucleotides that Induce Specific Binding of Aminoacyl-tRNAs to Ribosomes
isolated E. coli ribosomes bind a specific aminoacyl-tRNA in the presence of the corresponding synthetic polynucleotide messenger
a Each number represents the factor by which the amount of bound 14C increased when the indicated trinucleotide was present, relative to a control with no trinucleotide.
What is a complementary approach to possible codons?
Khorana used defined mRNAs in planned patterns to produce polypeptides with repeating patterns
(AC)n (alternating ACA and CAC codons) produced a polypeptide with equal amounts of threonine and histidine.
Given that a histidine codon has one A and two Cs, CAC must code for histidine and ACA for threonine.
Consolidation of the results from many experiments permitted assignment of 61 of the 64 possible codons
What is the effect of a termination codon?
three codons disrupted amino acid coding patterns when they occurred in a synthetic RNA polymer
synthesized dipeptides or tripeptides
these codons were identified as termination codons
What are some important things to know about amino acid code words in mRNAs?
61 codons code for amino acids
3 codons (UAA, UGA, UAG) are termination codons
1 codon (AUG) is the start / initiation codon (as well as the Met codon)
degenerate = an amino acid may be specified by more than one codon
The Genetic code is universal
exceptions include minor variations in mitochondria, some bacteria, and some single-celled eukaryotes
How is protein synthesis initiated?
initiation of protein synthesis in the cell relies on initiation codons and other signals in the mRNA
in retrospect, the experiments to identify codon function should not have worked without initiation codons
experimental conditions caused the initiation requirements for protein synthesis to be relaxed
What is open reading frame?
(protein synthesis; initiation)
open reading frame (ORF) = a reading frame without a termination codon among 50+ consecutive codons
long ORFs usually correspond to genes that encode proteins
a typical protein requires an ORF with 500+ codons
What is an anti-codon?
anticodon = a three-base sequence on the tRNA that base pairs with mRNA codons
base pairing occurs via hydrogen bonding
the alignment of the two RNA segments is antiparallel
What does a Wobble allow some tRNAs to recognize?
Wobble Allows Some tRNAs to Recognize More than One Codon
when several different codons specify one amino acid, the difference usually lies at the third base position
for example, alanine is encoded by GCU, GCC, GCA, and GCG
the third position in each codon is much less specific and is said to “wobble”
this allows certain tRNAs to recognize more than one codon
What is the wobble hypothesis?
the wobble hypothesis = proposes the third base of most codons pairs loosely with the corresponding anticodon base
permits rapid dissociation of the tRNA from its codon during protein synthesis
the first two bases of the codon form strong Watson-Crick base pairs with the anticodon
confers most of the coding specificity
What are relationships of the wobble hypothesis?
the first base of the anticodon (read in the 5′⟶3′ direction) determines the number of codons recognized by the tRNA
when an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs
at least 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids, 1 for initiation)
Inosinate (I) can form weak hydrogen bonds with A, U, and C
How many numbers of tRNAs are in E.coli cells?
most cells have more tRNAs than the 32 tRNAs required to translate all codons
The bacteriumE. colihas 47 different tRNA genes.
Many of these are present in multiple copies, such that there are 86 total tRNA genes in theE. coligenome.
What is a missense mutation?
missense mutations = mutations in which a single new base pair replaces another
most common type of mutation
in the third position of the codon, single base substitutions changes the amino acid only ~25% of the time
Two types: Transition and transversion
Transition: Purine replaced by purine [or pyri by pyri] (most common)
Transversion: Purine replaced by pyrimidine or vice versa (rare)
What is a translational frameshift?
translational frameshifting = “hiccupping” of ribosomes at a certain point in the translation to change the reading frame
allows 2+ related but distinct proteins to be produced from a single transcript
occurs during translation for the overlapping gag and pol genes of the retrovirus Rous sarcoma virus
What is RNA editing? (of mRNAs)
RNA editing = the addition, deletion, or alteration of RNA nucleotides in a manner that affects the meaning of the transcript during translation
addition or deletion of nucleotides is common in RNAs from mitochondrial and chloroplast genomes
Cytochrome Oxidase Subunit II: posttranscriptional editing inserts four U residues
-revises the reading frame
guide RNAs (gRNAs) = act as templates for the editing process
How is RNA editing altered by nucleotides?
commonly involves enzymatic deamination of adenosine or cytidine to form inosine or uridine, respectively
ADARs (adenosine deaminases that act on RNA) = carry out adenosine deamination reactions
APOBEC (apoB mRNA editing catalytic peptide) family of enzymes = carry out cytidine deaminations
includes AID (activation-induced deaminase) enzymes
What is RNA Editing of the Gene Transcript for the apoB-100 Component of LDL?
an APOBEC cytidine deaminase found only in the intestine binds to the apoB-100 mRNA and changes a Gln codon to a stop codon
produces a truncated protein
reaction permits tissue-specific synthesis of two different proteins from one gene
Give an overview of the 5 stages of protein synthesis.
- activation
- Initiation
- Elongation
- Termination
- Protein folding
What makes a ribosome a complex supramolecular machine?
each E. coli cell contains 15,000+ ribosomes
~25% of the dry weight of bacteria
bacterial ribosomes:
contain ~65% rRNA (forms the core and catalyzes peptide bond formation) and ~35% protein
have two unequal subunits (30S and 50S)
S (Svedberg unit) values = sedimentation coefficients that refer to their rate of sedimentation in a centrifuge
30S= 1 (16S rRNA)
50S= 2 (5S and 23S rRNAs)
What are the bacterial ribosomal subunits?
bacterial ribosomes have 30S and 50S subunits
-the combined sedimentation coefficient is 70S
50S and 30S subunits come together to form a cleft through which mRNA passes
there is no protein within 18 Å of the active site for peptide bond formation
high-resolution structure confirms that the ribosome is a ribozyme
Large subunit: 5S, 23S (Ribozyme)
Small subunit: 16S
What is the eukaryotic ribosomal structure?
eukaryotic ribosomes have a similar structure to bacterial ribosomes with somewhat increased complexity
eukaryotic ribosomes:
are larger (~80S) and more complex than bacterial ribosomes
have two subunits (60S and 40S on average)
Large subunit: 5S, 5.8S, 28S (probable role as Ribozyme)
Small subunit: 18S
Whats a big comparison between eukaryotic and prokaryotic ribosomes?
chloroplasts and mitochondria have simpler ribosomes than bacteria
How are the secondary structure in small subunit rRNAs conserved?
folding patterns of rRNAs are highly conserved in all organisms
How are ribosomes assembled in eukaryotes?
ribosomes are assembled through a hierarchical incorporation of r-proteins as the rRNAs are synthesized
processing of pre-rRNAs occurs within large ribonucleoprotein complexes
dozens of proteins and snoRNAs are involved in final maturation of the ribosome
What characteristic structural features do transfer RNAs have?
in both bacteria and eukaryotes, tRNAs are small and consist of 73-93 nucleotide ssRNA
mitochondria and chloroplasts contain smaller tRNAs
common structural elements:
8+ residues have modified bases and sugars (many are methylated derivatives)
have a guanylate (pG) residue at the 5′ end
have the trinucleotide sequence CCA(3′) at the 3′ end
have a cloverleaf shape in 2-D and a twisted L shape in 3-D
What is the amino acid arm of tRNA?
amino acid arm = carries a specific amino acid esterified by its carboxyl group to the 2′-OH or 3′-OH group of the A residue at the 3′ end of the tRNA
What is the anit-codon arm of tRNA?
anticodon arm = contains the anticodon
What is the D arm of tRNA?
D arm = contains the unusual nucleotide dihydrouridine (D)
contributes to overall folding of tRNAs
What is the TψC arm of tRNA?
TψC arm = contains ribothymidine and pseudouridine (ψ)
ψ has an unusual carbon-carbon bond between the base and ribose
contributes to overall folding of tRNAs
interacts with the large-subunit rRNA
How does Aminoacyl-tRNA Synthetases attach the Correct Amino Acids to their tRNAs?
stage 1:
occurs in the cytosol
activates the carboxyl group of each amino acid
establishes a link between each new amino acid and the information encoding it in the mRNA
tRNAs are “charged” when attached to their amino acid (aminoacylated)
What is aminoacyl-tRNA synthetases and what is its function?
aminoacyl-tRNA synthetases = esterify the 20 amino acids to their corresponding tRNAs
specific for one amino acid and 1+ corresponding tRNAs
most organisms have one enzyme for each amino
*aminoacyl-tRNA synthetases fall into Class I or Class II
no evidence of a common ancestor
based on differences in:
primary and tertiary structure
reaction mechanism
What is the first step in Aminoacyl-tRNA Synthetases?
the reaction catalyzed by an aminoacyl-tRNA synthetase is
amino acid + tRNA + ATP –>(mg2+) aminoacyl-tRNA + AMP + PPi
step 1 forms the enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP)
What is step 2 in the Aminoacyl-tRNA Synthetase Reaction?
step 2 transfers the aminoacyl group from enzyme-bound aminoacyl-AMP to its corresponding specific tRNA
mechanism depends on the enzyme class
In Aminoacyl-tRNA synthetase, how is the ester linkage affected? (think free energy)
(The Ester Linkage has a Highly Negative ∆G′°)
pyrophosphate formed in the activation reaction undergoes hydrolysis to phosphate by inorganic pyrophosphatase
two high-energy phosphate bonds are ultimately expended for each amino acid molecule activated
the overall reaction for amino acid activation is essentially irreversible:
amino acid + tRNA + ATP-->(Mg2+) aminoacyl-tRNA + AMP + PPi ∆G′° = −29 kJ/mol
What is the proofreading function of aminoacyl-tRNA?
Ile-tRNA synthetase has a proofreading function to distinguish between Val and Ile by a factor of 200.
Val-AMP fits into the hydrolytic (proofreading) site of Ile-tRNA synthetase
Val-AMP is hydrolyzed
tRNA does not become aminoacylated to the wrong amino acid
What are additional methods for enhancing fidelity of protein synthesis?
most aminoacyl-tRNA synthetases can hydrolyze the ester linkage in the aminoacyl-tRNAs
some aminoacylation active sites can sufficiently discriminate between the proper substrate and incorrect amino acids
The few aminoacyl-tRNA synthetases that activate amino acids with no close structural relatives (Cys-tRNA synthetase, for example) demonstrate little or no proofreading activity;
in these cases, the active site for aminoacylation can sufficiently discriminate between the proper substrate and any incorrect amino acid
What is the error rate of protein synthesis?
the overall error rate of protein synthesis is ∼1 mistake per 10^4 amino acids incorporated
higher than that of DNA replication
flaws in a protein have less biological significance
eliminated when the protein is degraded
not passed on to future generations
What is the “second genetic code”?
individual aminoacyl-tRNA synthetases must be specific for a single amino acid and for certain tRNAs
“second genetic code” refers to the interaction between aminoacyl-tRNA synthetases and tRNAs
nucleotides in tRNA that confer binding specificity are concentrated in:
the amino acid arm
the anticodon arm
the nucleotides of the anticodon itself
How is tRNA recognized by Ala-tRNA synthetases?
a single G=U base pair in the amino acid arm of tRNAAla determines tRNA recognition by Ala-tRNA synthetases
true across a range of organisms, from bacteria to humans
Ten or more specific nucleotides may be involved in recognition of a tRNA by its specific aminoacyl-tRNA synthetase. But in a few cases the recognition mechanism is quite simple.
What are two unusual amino acids in peptides?
selenocysteine = formed after charging a UGA-recognizing tRNA with serine in both bacteria and eukaryotes
pyrrolysine = directly attached to its tRNA that recognizes UAG (stop) codon in some archaea
There are actually 22 rather than 20 amino acids specified by the known genetic code. The two extra amino acids are selenocysteine and pyrrolysine, each found in only very few proteins but both offering a glimpse into the intricacies of code evolution. Formate dehydrogenase in bacteria and glutathione peroxidase in mammals require selenocysteine for their activity. Pyrrolysine is found in a group of anaerobic archaea called methanogens.
What is stage 2 in protein synthesis?
A Specific Amino Acid Initiates Protein Synthesis
*the AUG initiation codon specifies an amino-terminal methionine residue.
all organisms have two tRNAs for methionine:
one for when (5′)AUG is the initiation codon
one when a Met residue in an internal position in a polypeptide
How is the initiation complex formed in bacteria? (step 1)
IF3 prevents the 30S and 50S subunits from combining prematurely
Shine-Dalgarno sequence = region of mRNA that guides the initiating (5′)AUG to its correct position
the mRNA is complementary to a sequence in the rRNA
How is the initiation complex in bacteria formed? steps 2 and 3
the anticodon of fMet-tRNA(fMet) is correctly paired with the mRNA’s initiation codon at the P site
the initiation complex = a functional 70S ribosome containing mRNA and the initiating fMet-tRNA(fMet)
Whats the first step in the initiation of protein synthesis in eukaryotic cells?
eukaryotic mRNAs are bound to the ribosome as a complex with specific binding proteins
eukaryotic cells have at least 12 initiation factors
eIF1A is a functional homolog of IF1
eIF3 is a functional homolog of IF3
eIF1A blocks tRNA binding to the A site
eIF3 prevents premature joining of the ribosomal subunits
eIF1 binds to the E site
What is step 2 in the protein synthesis in eukaryotic cells?
a complex containing charged tRNAMet, eIF2, and GTP binds to the 40S subunit, along with eIF5 and eIF5B
–creates a 43S preinitiation complex
What is step 3 in the initiation of protein synthesis in eukaryotic cells?
-the eIF4F complex contains:
eIF4E (binds 5′ cap)
eIF4A (an ATPase and RNA helicase)
eIF4G (a linker protein)
-mRNA binds to eIF4F
mediates its association with the 43S preinitiation complex
(The eIF4G protein binds to eIF3 and eIF4E to provide the first link between the 43S preinitiation complex and the mRNA. The eIF4G also binds to the poly(A) binding protein (PABP) at theend of the mRNA, circularizing the mRNA (Fig. 27-27) and facilitating the translational regulation of gene expression)
How is mRNA circularized in the eukaryotic initiation complex?
eIF4G binds to the poly(A) binding protein (PABP) at the 3′ end of the mRNA, circularizing the mRNA
facilitating the translational regulation of gene expression
What is step 4 in the initiation of protein synthesis in eukaryotic cells?
addition of the mRNA and associated factors creates a 48S complex
the 48S complex scans the mRNA until AUG is encountered
**may be facilitated by the RNA helicase of eIF4A
What is step 5 in the initiation of protein synthesis in eukaryotic cells?
the 60S subunit associates with the complex
eIF5 promotes eIF2 GTPase activity
eIF2-GDP complex has reduced affinity for the initiator tRNA
eIF5B hydrolyzes GTP and triggers dissociation of eIF2-GDP and other factors
homologous to IF2
What is the elongation step 1?
Binding of an Incoming Aminoacyl-tRNA
an incoming aminoacyl-tRNA binds an GTP-bound EF-Tu complex
the aminoacyl-tRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex
after GTP hydrolysis, EF-Tu–GDP complex leaves the ribosome
How is the first peptide bond in bacteria made?
In the p-site
What is the ribosome rescue system?
Ribosome Pausing, Arrest, and Rescue
ribosomes may stall if mRNA is damaged or incomplete
tmRNA (transfer messenger RNA) rescues stalled bacterial ribosomes in a multistep pathway
permits release and degradation of the damaged mRNA
tags the truncated polypeptide for degradation
(In virtually all bacteria, the rescue system consists oftransfer-messenger RNA (tmRNA)andsmall protein B (SmpB). These bind to the stalled complex in such a way that the tmRNA is positioned in the empty A site so that the ribosome can continue translation until it encounters a stop codon embedded in the tmRNA. The ribosome is then recycled, and both the defective mRNA and the polypeptide translated from it are degraded. Similar systems exist in eukaryotes.)
How is protein synthesis terminated in bacteria?
ribosome recycling factor (RRF) and energy from GTP hydrolysis aid in ribosome dissociation
IF3 promotes dissociation of the tRNA
the complex of IF3 and the 30S subunit is then ready to initiate another round
What are Chaperonins in protein folding?
How does Farnesylation of a Cys residue happen?
slide 100 What is puromycin?
Protein Synthesis Is Inhibited by Many Antibiotics and Toxins
puromycin = an inhibitory antibiotic that binds to the A site and terminates polypeptide synthesis
made by the mold Streptomyces alboniger
structure similar to the 3′ end of an aminoacyl-tRNA
producing peptidylpuromycin
How are eukaryotic proteins directed with the appropriate signals to the er?
What key role does Glycosylation play in protein targeting?
N-glycosylation occurs in the in the ER lumen
involves a 14-residue core oligosaccharide that is built up stepwise
A few proteins areO-glycosylated in the ER, but mostO-glycosylation occurs in the Golgi complex or in the cytosol (for proteins that do not enter the ER)
(Picture=Synthesis of the Core Oligosaccharide of Glycoproteins)
How do proteins travel?
Proteins Destined for Lysosomes, the Plasma Membrane, or Secretion
proteins travel from the ER to the cis side of the Golgi complex in transport vesicles
sorting occurs in the trans side of the Golgi complex
How does phosphorylation of Mannose residues on lysosome-targeted enzymes happen?
N-acetylglucosamine phosphotransferase recognizes an unknown structural feature of hydrolases destined for lysosomes
What are targets of Nuclear proteins?
. A protein with an appropriate nuclear localization signal (NLS) is bound by a complex of importins α and β. 2. The resulting complex binds to a nuclear pore and translocates. 3. Inside the nucleus, dissociation of importin β is promoted by the binding of Ran-GTP. 4. Importin α binds to Ran-GTP and CAS (cellular apoptosis susceptibility protein), releasing the nuclear protein. 5. Importins α and β and CAS are transported out of the nucleus and recycled. They are released in the cytosol when Ran hydrolyzes its bound GTP. 6. Ran-GDP is bound by NTF2, and transported back into the nucleus. 7. Ran-GEF promotes the exchange of GDP for GTP in the nucleus, and Ran-GTP is ready to process another NLS-bearing protein-importin complex.
(b) Transmission electron micrograph of a freeze-fractured nucleus, showing numerous nuclear pores. The nuclear pore complex is one of the largest molecular aggregates in the cell . It is made up of multiple copies of more than 30 different proteins.
What is the model for protein export in bacteria?
SecB = chaperone protein
SecA = a receptor and a translocating ATPase
translocation complex = made up of Sec Y, E, and G
How do cells import proteins?
Cells Import Proteins by Receptor-Mediated Endocytosis
How are proteins degraded in eukaryotes?
ubiquitin = protein that is covalently linked to proteins slated for destruction via an ATP-dependent pathway
highly conserved protein
the ATP-dependent pathway includes three enzyme types:
E1 activating enzymes
E2 conjugating enzymes
E3 ligases
What is the 26S proteasome?
26S proteasome = a large complex that degrades ubiquitinated proteins
highly conserved
(before entering, ubiquitin is removed)
The outer rings are formed from sevenαsubunits, and the inner rings from sevenβsubunits. Three of theβsubunits have protease activities, each with different substrate specificity.
What is a simple signal for Ubiquitination?
the identity of the first residue has a profound influence on half-life
(all depends on where they are located)
