Protein Translation Flashcards
RNA species involved in gene expression and roles of each
- pre-mRNA
- snRNA makes mRNA
- mRNA moves out of nucleus
- rRNA
- tRNA
- miRNA
Role of mRNA cap in translation
7meG cap added on 5’ end (methyl guanicine)
Role of polyA tail and PABP in translation
150-200 adenosine residues added on
PABP: polyA binding protein interacts with polyA tail and cap binding complex (eIF4F)
Drastically improves translation of mRNA
Because once the ribosome comes to the termination codon, it will dissociate from mRNA but is now in close proximity to the cap binding complex so it can re-initiate
Contribute to circularization of ribosome bound mRNA
Steps of initiation in translation
- Initial recognition
Cap binding complex (eIF4F) binds mRNA at the 7meG cap (which is on the 5’ mRNA)
Initiating complex assembles on cap binding complex - eIF2 loads methionyl-tRNA onto small ribosomal unit (met-tRNA initiates ~95% of translation)
- Small ribo subunit is recruited to mRNA
eIF1 and eIF3 mediate interaction between small ribo subunit and the cap binding complex (eIF4F) on mRNA cap - Small ribo subunit scans along mRNA
- Recognition of start codon (AUG/met- 95% of time); release of initiation factors; met can also be used elsewhere in protein
- Binding of large ribo subunit (elongation commences); now tRNA can inter the ribosome and translation can initiate
Role of elongation factors in translation
A site: aminoacyl site; incoming aa-tRNA binds here
P site: peptidyl site for growing peptide chain attached to tRNA; holds tRNA bound to nascent protein
E site: empty tRNA moves from P to E site; “naked” tRNA binds here after its aa is added to protein
EF1: Elongation factor 1 “escorts” next aa-tRNA to the now vacant A site
A G protein that hydrolyzes one GTP for addition of AA.
EF2: Translocation of ribosome on mRNA requires activity of this elongation factor
A G protein that hydrolyzes one GTP every time the ribosome moves a codon length
Role of termination factors in translation
When the ribosome reaches the stop codon (UAG, UAA, UGA), a release factor (RF) binds to A site…
- Catalyzes release of protein from tRNA
- Ribosome disassembles
- Ribosomal subunits are recycled
Toxins that inhibit elongation
Toxins target 28s rRNA by cleaving adenine base off sugar
-Damages ribosome
-Damages RNA
-Loss of protein synthesis
Ex. Some bacteria (shiga toxin) and
Castor beans (ricin toxin)
Same mechanism, shows conversion evolution
Toxins that inhibit EF2
Diptheria toxin and Exotoxin A
Catalyzes ADP ribosylation of EF2, blocking GTP hydrolysis
-Shuts down all translation
-Among the most toxic proteins, very teeny amounts are lethal
Cap binding complex
eIF4F
F = A + G + E (comprised of three proteins)
Adaptor between cap and the ribosome
- Ensures only mRNA is being translated
- Recruit ribosome and unwind secondary structure in mRNA
- Binding can control translation
Limiting subunit of the cap binding complex (eIF4F)
eIF4E
Binds the 7meG cap
miRNA
Short 22-23 nucleotide RNA
miRNA base pairs with mRNA
1. Directly leads to repression of translation of target mRNA and 2. Recruits machinery to degrade target mRNA
~affecting stability of RNA~
A way to silence production of protein from that message
Called post-transcriptional gene silencing (PTGS) or when used experimentally, RNA interference (RNAi)
tRNA
Serve as adaptors between nucleotide sequence of mRNA and the AA being inserted
codon 5-3
anti codon of tRNA 3-5
“Acceptor Stem”
Ester bond:
Between adenosine of tRNA and AA
High energy bond which is utilized for the formation of the peptide bond in protein synthesis
Molecule:
- Modified nucleotides
- Stem loop structure
- Intramolecular base pairing
Diseases can be caused by mechanisms involved in tRNA modifications and mutations
Peptide Bond Formation
Catalyzed by peptidyl transferase
Large ribosomal subunit is a catalytic molecule NOT a protein
Covalent bond formed between the:
Amino group (A site)
C of carboxylic group (P site)
Formation results in shift of nascent peptide from the P site to A site, then the ribosome translocates to next codon, shifting the peptide back to the P site
Cap binding complex
eIF4F
F = A + G + E (comprised of three proteins)
Adaptor between 7meG cap on mRNA and the ribosome
- Ensures only mRNA is being translated
- Recruit ribosome and unwind secondary structure in mRNA
- Binding can control translation
Limiting subunit of the cap binding complex (eIF4F)
eIF4E: Binds the 7meG cap
One way to regulate transcription by broad general factors: target the limiting subunit
Controlling the availability of eIF4E will broadly affect translation
4E binding proteins (4E-BPs) bind eIF4E
No insulin:
4E-BP is not phosphorylated, allowing it to interact with CAP binding complex, taking the binding complex out of circulation, inhibiting its ability to bind with mRNA, reducing translation
Insulin:
Pro growth, pro metabolic
Insulin > Receptor > Kinase cascade > Phosphorylation of 4E-BP1
Phosphorylation of 4E-BP takes it out of circulation, freeing up the CAP binding complex so it can bind to mRNA and increase translation
Translation by cellular stress (unfolded protein response UPR)
- Unfolded protein response
2. ER stress
Mechanisms of Translational Regulation (2)
- General factors (eIF) are broad/global
2. microRNA (miRNA) are specific
Translation by pro-growth signaling
eIF4E limiting subunit is binding on the cap is inhibited by 4E-BP
Mechanisms of Translational Regulation (2)
1. General factors (eIF) are broad/global eIF2 phosphorylation (shuts off translation except pro-recovery genes) - unfolded protein response eIF4E (limiting subunit) binding inhibited by 4E-BPs
- microRNA (miRNA) are specific
Initiation/general factors, eIF2a phosphorylation
One mechanism of translational regulation that is global
When eIF2a gets phosphorylated, all of translation shuts down with few exceptions
{IF2a can no longer participate in initiation, it’s taken out of the equation; the cell can no longer make a complex between eIF2, met-tRNA and the small ribosomal subunit}
Four kinases that phosphorylate eIF2a, inhibiting translation:
- PKR
- GCN5
- HRI: activated by low heme only in immature erythrocytes; not enough heme being made for globin; HRI activation shuts down globin protein translation
- PERK: activated by ER stress/unfolded proteins
miRNA vs siRNA
siRNA: (small, interfering) delivered as a dsRNA "drug", processed in cytoplasm Targets RISC to mRNAs Induce mRNA degradation NO DIRECT EFFECT on translation **typically drugs being tested**
miRNA: (micro) produced inside cell, processed by RNA Pol II in nucleus
Targets RISC to mRNAs
Decrease translation
Induce mRNA degradation
Medical application of miRNA
miRNA
- Involved in development/function of all cell types/organ systems
- Dysregulation of specific miRNAs associated with many disease
- Expression of processing enzymes can be altered in disease
- Useful as biomarkers for tumor burden/effectiveness of treatment
Therapeutic approach:
- Silence expression of protein required for pathology
- dsRNA oligonucleotides delivered as “drug”
- called RNAi (interference) in this context
Anti-bacterial agents
~50% of antibiotics inhibit translation of bacteria (bacteria synthesis)
Antibiotics can
- Inhibit bacterial translation, useful for killing bacteria
- Immediately shut off production of additional bacterial toxin
Aminoglycosides (-mycins) have multiple mechanisms
- Block initiation
- Block further translation and elicits premature termination (translocation)
- Incorporation of incorrect AA
Frameshift mutation
Insert or loss of nucleotides in the ORF that is not a multiple of three
Random AA will be incorporated
Protein will not fold properly, will not function
STOP codon quickly encountered
Truncated protein quickly degraded
Missense mutation
A single nucleotide substitution that can result in the incorrect AA incorporated into a protein
Ex. Mutation of h-RAS in a bladder cancer cell line
Sufficient to make h-RAS “always on” gain of function mutation
Nonsense mutation
Creation of premature stop codon by changing one or more nucleotides
Non-functional, truncated protein
Silent mutation and SNPs
When nucleotide substitutions do not result in a different AA incorporated
Particularly at the wobble position
Possible because of degeneracy
SNPs: Single nucleotide differences scattered across the genome that represent the genetic differences among individuals at a given position
Effort is placed on identifying SNPs that contribute to disease susceptibility
non-synonymous: SNP results in a AA change vs synonymous
Three types of mRNA surveillance associated with the first round of protein translation and conditions under which leads to mRNA decay and loss of protein expression from a mutated gene
Nonsense-mediated decay: [frameshift]
When the premature termination codon is at least ~50 nucleotides upstream from final exon-exon junction
Trigger mechanisms that lead to rapid destruction of mRNA
Loss of all mRNA and protein made from that gene
No-go decay:
When translation ribosome pauses too long on mRNA, result of stem loop structure on mRNA.
Non-stop decay:
When mRNA through mutation or splicing error, lacks a stop codon
Acetylation
Only occurs on lysine
Phosphorylation of AA
ONly happens on serine, threonine and tyrosine
Mechanism in Parkinson’s, Lewy Body Dementia, Huntington’s Disease, Alzheimer’s
Neurodegenerative diseases with unfolded proteins
UPR is chronically activated
Thought to lead to neuronal dysfunction and death due to
- Inhibited protein synthesis
- Activation of apoptotic pathways through UPR mechanisms
Intrinsically disordered sequences
Rich in Pro and Gly
pH sensor AA
Histidine because pKa is close to normal (6.1)
