RNA to Protein Flashcards
The study of the mechanisms of transcription and translation
Molecular biology
Protein synthesis is very important in part because it is the last opportunity for
Regulating gene expression
The genetic code is degenerate, with the exception that
The UGA stop codon encodes selenocysteine in humans
Mutation that changes the codon to a termination codon
Nonsense mutation
Mutation that changes the codon to another sequence coding the same amino acid
Silent muttion
Mutation that changes the codon to a sequence that encodes an entirely different amino acid
Missense mutation
What are the six features of a typical mammalian mRNA?
- ) 5’ Cap
- ) 5’ UTR
- ) Start codon (AUG)
- ) Stop codon (UAA)
- ) 3’ UTR
- ) Poly A tail
The region between the start and stop codon
Coding region
Specialized nucleotide (7-methyl-GMP) that is required for binding of initiation factors
5’ cap
The start codon AUG codes for
Methionine
What are the three stop codons
UGA, UAG, and UAA
The sequence between the stop codon and the poly(A) tail, and the site of key regulatory sequences
3’ UTR
Protects mRNA from degredation and increases translational efficiency
-Un-templted
Poly(A) tail
Deletion of a base can be disastrous because it will change the
Reading frame
The 3’ CCA end of tRNA is not trancribed, but is added after processing and it
Attaches to amino acids
Recognize tRNAs and add the appropriate amino acid to them
aminoacyl tRNA synthetases
Aminoacylation requires
ATP
Recognize the anticodon as well as other structural features in the tRNA
aminoacyl tRNA synthetases
The eukaryotic ribosome (80S) is made up of
- ) A large subunit (60S)
2. ) A small subunit (40S)
The ribosome is mostly RNA. Peptide bond formation occurs without the contribution of any protein, meaning the ribosome is a
ribozyme
Fill the gap between the large and small subunits of the ribosome
tRNAs
What are the three tRNA binding sites in a ribosome?
- ) A (aminoacyl) site: initial tRNA binding site for next codon
- ) P (peptidyl) site: where peptide bond is formed
- ) E (exit) site: tRNA is released
Initiation and elongation occur simutaneously, yielding multiple ribosomes on a single mRNA molecule. This very large mRNA/ribosome complex is called a
Polysome
Encompasses the assembly of the ribosome and mRNA and the positioning of the ribosome on the start codon
Initiation
The first step of initiation is the formation of the
Pre-initiation complex (made up of elongation factors like GTP dependant elF2)
The second step of initiation is the pre-initiation complex
Sans the mrNA for the AUG codon
Part of the initiation complex that binds to the 5’ cap
elF4E
Part of the initiation complex the binds and delivers initiator Met-tRNA
-requires GTP
elF2
PArt of initiation complex that serves as a scaffold protein that binds elF4E required for assembly of the pre-initiation complex
elF4G
The initiator tRNA is special in that it is not used during
Elongation or to incorporate Met
The initiator tRNA moves along the RNA searching for the first start codon. During this process, it utilizes
Helicase activity and ATP hydrolysis
The third step of initiation is that after the start codon has been located, the
Large subunit joins the complex
Causes elF2 release, signalling the large ribosomal subunit to bind
GTP hydrolysis
A common theme in many steps of translation. It often provides a signal for the next step (i.e. the binding of the 60S subunit)
GTP-dependent release of factors
The movement of the ribosome down the mRNA, coordinated with aminoacyl tRNA delivery
Elongation
During initiation, the very first tRNA is bound in the P site. All subsequent tRNAs first bind to the
A site
What is step one of elongation
Delivery of aa-tRNA to the A site and E site release
The incoming tRNA is bound to the GTP-dependent factor
eEF1A
What is step two of elongation?
GTP hydrolysis and eEF1A release, followed by proofreading
What is step three of elongation?
eEF2 (w/ GTP) binding to catalyze translocation
What is step four of elongation?
GTP hydrolysis and eEF2 release (completion of cycle)
Binds to all canonical tRNA’s (i.e. all tRNAs except the initiator tRNA and the selenocysteine tRNA
eEF1A
A G-protein that is required for ribosome translocation
eEF2
Occurs in the ribosomal A-site, where codon/anticodon pairs are “checked” by ribosome conformation
Proofreading
Is there a terminator tRNA?
No
A tRNA mimetic that catalyzes the release of the completed peptide, signalling termination
eRF1 (enzyme release factor 1)
Unlike in eukaryotes, bacterial transcription and translation are
Coupled
Bacterial mRNAs are different from eukaryotic mRNAs in that they are
Polycystronic (a single code encodes multiple proteins)
In bacteria, initiation is dictated by a sequence upstream of the start codon, which directly base pairs to the ribosomal RNA. This is called the
Shine-Dalgarno sequence
Bacterial ribosomes are smaller and sufficiently divergent from mammalian ribosomes, to allow
Selective inhibition by the ribosome inhibitor class of antibiotics
Typically do have residual toxicity due to their effects on mitochondrial ribosomes, which are more similar to bacterial ribosomes
Ribosome inhibitors
What are the sedimentation coefficients for the large and small ribosomal subunits of the prokaryotic ribosome?
30S and 50S for a 70S ribosome
What are the prokaryotic counterparts for the following eukaryotic enzymes?
- ) eIF2
- ) eEF1A
- ) eEF2
- ) IF2
- ) EFTu
- ) EFG
Post transcriptional, but pre-translational, regulation
mRNA editing
The apolipoprotein B is made in the liver and intestine. However, the intestine requires a shorter version. Thus
apoB mRNA is edited to induce a premature stop codon for the intestinal apoB
Short RNA molecules that base pair with mRNAs and regulate translation
micro RNAs (miRNAs)
miRNAs usually bind to the 3’ UTR and
inhibit translation
A protein complex thought to physically impede translation initiation
RISC
Many miRNAs are correlated with
Diseases
Iron homeostasis is regulated by an
-binds the iron regulatory element (IRE) and prevents translation
Iron sensing protein (IRP)
The IRP can not bind the IRE if it is bound to
+Fe2+
Some mRNAs encoding proteins that regulate iron homeostasis contain an “iron response element (IRE)” that binds to an iron-sensing protein (IRP) that regulates
Translation or mRNA stability depending on cellular ion concentrations
Nutritional status regulates
Translation
Phosphorylation of an initiation factor (eIF2) causes translation inhibition in response to
- ) Low amino acid concentration
- ) Cellular stress (e.g. oxidative stress)
- ) Immune response
- ) Unfolded proteins
To assist in re-binding GTP after hydrolysis, the G-protein eIF2 requires a
Guanine Nucleotide Exchange Factor (GEF) (which for eIF2 is eIF2B)
GEFs are required because the
Affinity for GDP is much higher than that for GTP
Under normal conditions, GDP is echanged for GTP by the action of eIF2B. However, poor nutrition (i.e. reduced amino acid concentrations) causes eIF2 phosphorylation, which
Causes eIF2 to bind eIF2B as an inactive complex.
What happens to protein synthesis when phosphorylated eIF2 binds eIF2B?
There will be an excess of inactive eIF2 (the GDP bound form), and protein synthesis slows dramatically
Hypoxia regulates
translation
The mTOR (mammalian Target Of Rapamycin) signaling pathway represses translation in response tohypoxia by regulating the function of
eIF4E
Is like a cellular rheostat - it upregulates
translation during growth and downregulates
during stress. It is constitutively in an
active state in order to keep growth in check.
The mTOR pathway
Normoxia (growth conditions) induces
mTOR signaling
Proteins that bind to eIF4E and inhibit translation
4EBPs
When 4EBPs are phosphorylated, they are
Released from eIF4E (ending inhibition)
The mTOR pathway is a key regulator of
4EBP phosphorylation
Activation of mTOR (during growth) causes
4EBP phosphorylation and increased translation
Inhibits mTOR, and thus allows 4EBP dephosphorylation and binding to eIF4E, causing decreased translation
Hypoxia
What is often called the 21st amino acid?
deficiency causes hyperthyroidism
Selenocysteine (Sec)
Selenocysteine incorporation requires alternative usage of the
UAG stop codon
The deiodinases are required for TH synthesis, thus among other things, selenium is essential for
Proper thyroid function
Selenocysteine incorporation requires a unique set of
Translation factors
A specialized elongation factor, similar to eEF1A,
that binds to the Sec-tRNA
eEFSec
A specialized tRNA that recognizes UGA codons and carries the selenocysteine amino acid
Sec-tRNA^Sec
An RNA element in the 3’ UTR required for Sec incorporation
SECIS element
Binds to the SECIS element and assists in getting eEFSec ternary complex to the ribosome at UGA Sec codons
SECIS binding protein 2 (SBP2)
Encoded by a UGA stop codon that has been “re-coded” to allow Sec-tRNA binding
Selenocystein
Required for the “re-coding” of the stop codon
SBP2
Caused by SBP2 mutations in humans because reduced Sec incorporation leads to reduced production of the deiodinases
Some rare forms of hypothyroidism
Creating more than one protein from a single mRNA
-Utilized by viruses like HIV
Ribosomal frameshifting
A single mRNA can encode more than one protein product if the ribosomes are signaled to
Frameshift
Frameshift signals include the
RNA pseudoknot, and an RNA sequence called the “slippery site”
The efficiency of frameshifting determines the amount of each protein that is
Made
Targets translation by modifying the elongation factor responsible for translocation (eEF2)
Diptheria toxin
Transcriptional regulation sometimes takes too long, for a faster response, we want
Translational regulation
Typically, a frame shift mutation will result in
Premature termination (Stop codon formation)
Typically, a ribozyme is an enzyme that can cleave
RNA
Place where you maintain the registry of the mRNA and maintain the reading frame. The actual function of the is still debated
E-Site
Most proteins in the translation process are
-Act as switches to sequence the events of translation
G-proteins (GTP dependent)
The pre-initiation complex comes across a lot of secondary structural motifs. In order to break these structures up, it relies on a
Helicase (Only step in protein synthesis that requires ATP)
Occurs in step 2 of elongation. If there are no errors, then we will get GTP hydrolysis and eEF1A release
Proofreading
Uses GTP hydrolysis to catalyzes the translocation mechanism that moves the new peptide from the A site to the P site
eEF2
eEF2 binds into the A site, which forces the A site tRNAs to move into the P site. GTP hydrolysis allows the release of eEF2 from the A site and the ribosome shifts by
One codon
Feedback on the initiation complex and physically block translation
RISC complex
Can not bind the IRE and thus, translation is normal and we produce ferratin
Iron-bound iron regulatory protein
Bind and inhibit eIF4E, the protein which binds to the cap to allow translation to occur
4E Binding Proteins (4EBP)
In children, deficiencies in selenium present as
Hypothyroidism
caused by SBP2 mutations in humans because reduced Sec incorporation leads to reduced production of the deiodinases
Some rare forms of hypothyroidism