Protein synthesis Flashcards
A researcher has discovered a temperature-sensitive cell line that displays an overall reduction in protein synthesis. Analysis of the mRNA produced at the nonpermissive temperature indicated that a key structural feature, normally present on mRNA, was missing. Such a structure is most likely which one of the following?
A. Intron-exon secondary structure
B. Pseudouridine
C. The 5’ cap
D. Thymine
E. The poly-A tail
C. The 5’ cap. The 5’ cap of mRNA is recognized by eIF4E to allow ribosome assembly on the mRNA. The absence of a cap would not allow a translation initiation complex to form. Factor eIF4e is required for the mRNA bining to the small ribosomal subunit through cap recognition.
Not A b/c introns are not found in mature mRNA, they are removed by splicing in the nucleus; thus intron-exon secondary structure wouldn’t be present.
Not B b/c pseudouridine is found only in tRNA, not mature mRNA.
Not D b/c Thymine, while found in tRNA by posttranscriptional processing, is not found in mRNA.
Not E b/c the poly-A tail at the 3’ end of the mRNA adds stability to the mRNA, but doesn’t play a role in translation initiation.
Initiation of translation usually involves the interaction of certain key proteins with a special tag bound to the 5’-end of an mRNA molecule, the 5’ cap. The protein factors bind the small ribosomal subunit (also referred to as the 40S subunit), and these initiation factors hold the mRNA in place. The eukaryotic Initiation Factor 3 (eIF3) is associated with the small ribosomal subunit, and plays a role in keeping the large ribosomal subunit from prematurely binding. eIF3 also interacts with the eIF4F complex which consists of three other initiation factors: eIF4A, eIF4E and eIF4G. eIF4G is a scaffolding protein which directly associates with both eIF3 and the other two components. eIF4E is the cap-binding protein. It is the rate-limiting step of cap-dependent initiation, and is often cleaved from the complex by some viral proteases to limit the cell’s ability to translate its own transcripts. This is a method of hijacking the host machinery in favor of the viral (cap-independent) messages. eIF4A is an ATP-dependent RNA helicase, which aids the ribosome in resolving certain secondary structures formed by the mRNA transcript.
There is another protein associated with the eIF4F complex called the Poly(A)-binding protein (PABP), which binds the poly-A tail of most eukaryotic mRNA molecules. This protein has been implicated in playing a role in circularization of the mRNA during translation.[1][2] This pre-initiation complex (43S subunit, or the 40S and tRNA) accompanied by the protein factors move along the mRNA chain towards its 3’-end, scanning for the ‘start’ codon (typically AUG) on the mRNA, which indicates where the mRNA will begin coding for the protein.
Cap-dependent initiation of eukaryotic transcription pt. 1
In eukaryotes and archaea, the amino acid encoded by the start codon is methionine. The initiator tRNA charged with Met forms part of the ribosomal complex and thus all proteins start with this amino acid (unless it is cleaved away by a protease in subsequent modifications). The Met-charged initiator tRNA is brought to the P-site of the small ribosomal subunit by eukaryotic Initiation Factor 2 (eIF2). It hydrolyzes GTP, and signals for the dissociation of several factors from the small ribosomal subunit which results in the association of the large subunit (or the 60S subunit). The complete ribosome (80S) then commences translation elongation, during which the sequence between the ‘start’ and ‘stop’ codons is translated from mRNA into an amino acid sequence—thus a protein is synthesized
Regulation of protein synthesis is dependent on phosphorylation of initiation factor eIF2 which is a part of the met-tRNAi complex. When large numbers of eIF2 are phosphorylated, protein synthesis is inhibited. This would occur if there is amino acid starvation or there has been a virus infection. However, naturally a small percentage is of this initiation factor is phosphorylated. Another regulator is 4EBP which binds to the initiation factor eIF4E found on the 5’ cap on mRNA stopping protein synthesis. To oppose the effects of the 4EBP, growth factors phosphorylate 4EBP, reducing its affinity for eIF4E and permitting protein synthesis
Cap-dependent initiation of eukaryotic transcription pt. 2
The best studied example of the cap-independent mode of translation initiation in eukaryotes is the Internal Ribosome Entry Site (IRES) approach. What differentiates cap-independent translation from cap-dependent translation is that cap-independent translation does not require the ribosome to start scanning from the 5’ end of the mRNA cap until the start codon. The ribosome can be trafficked to the start site by ITAFs (IRES trans-acting factors) bypassing the need to scan from the 5’ end of the untranslated region of the mRNA. This method of translation has been recently discovered, and has found to be important in conditions that require the translation of specific mRNAs, despite cellular stress or the inability to translate most mRNAs. Examples include factors responding to apoptosis, stress-induced responses
Cap-independent initiation of eukaryotic transcription
Elongation is dependent on eukaryotic elongation factors. At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis. The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA. During chain elongation, each additional amino acid is added to the nascent polypeptide chain in a three-step microcycle. The steps in this microcycle are (1) positioning the correct aminoacyl-tRNA in the A site of the ribosome, (2) forming the peptide bond and (3) shifting the mRNA by one codon relative to the ribosome.
Translation can also be affected by ribosomal pausing which can trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. Ribosomal pausing also aids co-translational folding of the nascent polypeptide on the ribosome, and delays protein translation while its encoding mRNA. This can trigger ribosomal frameshifting.[4]
Eukaryotic translation: elongation pt. 1
Elongation in eukaryotes is carried out with two elongation factors: eEF-1 and eEF-2.
The first is eEF-1, and has two subunits, α and βγ. α acts as counterpart to prokaryotic EF-Tu, mediating the entry of the aminoacyl tRNA into a free site of the ribosome. βγ acts as counterpart to prokaryotic EF-Ts, serving as the guanine nucleotide exchange factor for α, catalyzing the release of GDP from α.
The second elongation factor is eEF-2, the counterpart to prokaryotic EF-G, catalyzing the translocation[disambiguation needed] of the tRNA and mRNA down the ribosome at the end of each round of polypeptide elongation.
Eukaryotic translation: elongation pt. 2
Eukaryotic translation termination factor 1 (eRF1), also known asTB3-1, is a protein that in humans is encoded by the ETF1 gene.[2][3][4]
In eukaryotes, there is only one release factor, eRF, which recognizes all three stop codons.
UGA
UAG
UAA
Eukaryotic translation: Termination
Under conditions of active exercise, protein synthesis is reduced in the muscle. Under these conditions, which aspect of translation is inhibited?
A. Inability to initiate translation
B. Inability to elongate during translation
C. Inability to terminate translation
D. Inability to synthesize mRNA
E. Inability to produce rRNA
A. Inability to initiate translation
As muscle works, and AMP levels, the muscle wants to preserve its ATP for muscle contraction, and not to use it for new protein synthesis. The increase in AMP levels lead to the activation of AMP-activated protein kinase, which will phosphorylate and inactivate eIF4E, which is a necessary component in recognizing the 5’ cap structure of the mRNA to allow ribosome assembly on the mRNA. The activation of the AMP-activated protein kinase does not alter elongation or the termination of translation. It does not block overall transcription, either of mRNA or rRNA, although it may lead to an inhibition of ribosomal biogenesis as well as the transcription of certain specific genes.
A young child exhibits the following symptoms: Coarse facial features, congenital hip dislocation, inguinal hernias, and severe developmental delay. These symptoms are fully evident at the child’s age of 1. Cellular analysis demonstrated the presence of inclusion bodies within the cytoplasm of liver cells. The inclusion bodies are the result of which of the following?
A. Enhanced lysosomal enzyme activity
B. Reduced lysosomal enzyme activity
C. Enhanced peroxisomal enzyme activity
D. Reduced peroxisomal enzyme activity
E. Enhanced protein secretion
B. reduced lysosomal enzyme activity. The child has I-cell disease (mucolipidosis type II), a deficiency in protein sorting, particularly of sending lysosomal enzymes to the lysosome. The I of I-cell disease stands for inclusion bodies. Lysosomal enzymes are tagged with mannose-6-phosphate (M6P) during posttranslational modification. Enzymes containing M6P to a M6P receptor, which transports the enzymes to the lysosomes. Lacking such a signal, patients with I-cell disease secrete their lysosomal contents into the plasma and interstitial fluids. This leads to lysosomal dysfunction and cellular destruction. Phosphotransferase is defective in patients with I-cell disease, so it doesn’t put the M6P on the lysosomal enymes so they aren’t sent to the lysosome.
A euk. cell line contains an aberrant, temperature-sensitive ribonucleases that specifically cleaves the large rRNA molecule into many pieces, destroying its secondary structure and its ability to bind to ribosomal proteins. This cell line, at the nonpermissive temperature, has greatly reduced the rates of protein synthesis. This rate-limiting step is which of the following?
A. Initiation
B. Termination
C. Elongation
D. Peptide bond formation
E. tRNA activation and charging
D. Peptide bond formation. It’s the large rRNA that catalyzes peptide bond formation, using peptides and amino acids in the “A” and “P” sites on the ribosome. A site =amino-acyl site. P site =peptidyl site. Destroying the secondary structure of this rRNA via the aberrant ribonuclease will limit the ability of the ribosome to create peptide bonds. The large, ribosomal RNA molecule is not essential for the initiation, termination, elongation (moving the ribosome along the mRNA after peptide bond formation has occurred), or tRNA and charging.
A cell contains a mutated alanine-tRNA ala synthetase that recognizes glycine instead of ala as its substrate. The anticodon of the tRNA recognized by this enzyme is IGC. When the cell translates the following portion of an mRNA molecule (presented in frame beginning with the 5’ nucleotide), what will be the amino acid sequence of the protein produced from this stretch of mRNA?
5’-AUG-GCG-GAC-UCG-GCU-AUG-3’
A. M-G-S-D-G-M
B. M-A-D-S-G-M
C. M-A-D-S-A-M
D. M-A-S-D-A-M
E. M-G-D-S-A-M
B. M-A-D-S-G-M. The variant cell line will mischarge a tRNA ala with glycine, but only the tRNAala that has, as its anticodon, IGC. IGC will recognize three codons: GCA, GCU, and GCC. Thus, when these codons are present in the mRNA, glycine will replace alanine. Thus, reading the RNA from the 5’ end (as translation reads the mRNA 5’ to 3’) provided in a series of 3, we have AUG (which is methionine), GCG (which is ala-the anticodon for this codon is CGC; ICG will not recognize the GCG codon), GAC (which is asp), UCG (which is Ser), GCU (which, in this cell line, is gly and not ala, since the IGC anticodon will recognize the GCU codon), and AUG (met);
A 3-yr-old boy, whose parents did not immunize him due to fears of postimmunization side effects, exhibited fever, chills, severe sore throat, lethargy, trouble breathing, and a husky voice. Physical exam indicated greatly enlarged lymph nodes, an increased heart rate, and swelling of the palate. A picture of the boy’s throat is shown below.
The throat is dull red, and a gray exudate (pseudomembrane) is present on the uvula, pharynx, and tongue.
A necessary cofactor for allowing these symptoms to appear in the child is which of the following?
A. ATP
B. NAD+
C. FAD
D. Acetyl-CoA
E. UDP-glucose
B. NAD+. Diptheria toxin, after entering cells, is cleaved by a protease to form an active enzyme, which, utilizing NAD+ as a substrate, ADP-ribosylates eEF2, thereby inhibiting protein translation. ATP, FAD, acetyl-CoA, and UDP-gluc are not required for the ADP-ribosylation rxn. The final modified product, an arginine with an ADP-ribose attached.
A 3-yr-old boy, whose parents did not immunize him due to fears of postimmunization side effects, exhibited fever, chills, severe sore throat, lethargy, trouble breathing, and a husky voice. Physical exam indicated greatly enlarged lymph nodes, an increased heart rate, and swelling of the palate. A picture of the boy’s throat is shown below.
The throat is dull red, and a gray exudate (pseudomembrane) is present on the uvula, pharynx, and tongue.
The molecular mechanism responsible for these physical observations in the boy is which of the following?
A. Activiation of protein kinase A
B. Activation of an elongation factor for translation
C. Glycosylation of a G protein
D. Inhibition of protein kinase A
E. Inhibition of an elongation factor for translation
E. Inhibition of an elongation factor for translation. The child has diptheria, which is caused by a bacterium (Corynebacterium diptheriae), which produces a toxin that leads to the inhibition of eEF2 which is required for the movement of tRNA from the A site to the P site. The toxin catalyzes the ADP-ribosylation (using NAD+ as a substrate) of eEF2 to bring about this inhibition. If one treats such a child with nicotinamide (the rxn product resulting from the loss of ADP-ribose from NAD+), one can reverse and block the ADP-ribosylation reaction catalyzed by the toxin. The toxin has no effect on protein kinase A, nor does it glycosylate a G protein. Diptheria causes sore throat, fever, swollen nodes (bull neck), weakness, hoarseness, painful swallowing and chills. The hallmark of the disease is a thick, gray membrane covering the pharynx.
