Translation Flashcards
What are the main feature of a mature mRNA? (x4)
- AUG - start codon
- UAG (AUU, UGA) - stop codon
- ORF - open reading framing
- UTR - untranslated region
What is AUG and where is it on the mRNA?
Start codon and it is at the 5’ end of mRNA
What is UAG, UAA, and UGA and where will these be located on the mRNA?
Stop codon. Located at the 3’ end and upstream of poly-A tail
What is the open reading frame of mRNA?
The region of mRNA that will be translated and encodes a protein
What is UTR on mRNA?
Untranslated region. Located right downstream of open reading frame
What are the features of tRNA? x2
- Acceptor arm (CCA)
- Anti-codon arm
What is the acceptor arm of tRNA?
Part of tRNA located at the 3’ end of tRNA and binds to incoming activated amino acid
What is the anti-codon arm of tRNA?
Anneals with the codon of mRNA. Contains a anticodon that is complimentary and antiparallel to codon in mRNA
the anticodon of the tRNA is ____ to the codon of mRNA
complimentary and antiparallel
When does amino acid activation occur and what is it?
Before translation.
Aminoacyl tRNA synthetase catalyzes attachment of amino acid to the 3’ end of tRNA and is important step for future formation of peptide bond
Aminoacyl tRNA sythestase
Used for amino acid activation. Catalyzes the attachment of an amino acid to the 3’ end of tRNA. Uses ATP and the high energy bond created is used for peptide bond linking. It can self check against incorrecpt pairing of an amino acid and tRNA
How many aminoacyl tRNA synthetases are there?
- 1 enzyme for each amino acid
What is interesting about the number of codons and tRNAs?
More codons than tRNA because multiple codons can code for an amino acid
What is the start codon and what does it code for?
AUG. Codes for methionine (Met)
What are two characteristics about codons when it comes to anti-codon pairing?
Unambiguous - single codon specifies one amino acid
Degenerate - many codons specify one amino acid
What do rRNAs form and where?
rRNAs combine with proteins in the nucleolus to form the small (40S) and large (60S) ribosomal subunits
What happens to the ribosomal subunits at the start of protein synthesis?
The two subunits join into 80S ribosome
What are the general initiation steps of translation?
Step 1: Assembly of the small ribosomal subunit, tRNA carrying Met, and mRNA. Guided by eukaryotic initiation factors (eIFs)
Step 2: Large subunit binds the complex
Guides the assembly of the small ribosomal subunit, tRNA carrying Met, and mRNA during initiation of translation
Eukaryotic initiation factors (eIFs)
Describe in detail the first step of inititiation of translation? (x5)
- eIF-2 binds GTP and activates
- activated eIF-2 binds met-tRNA (initiator tRNA) creating the ternary complex
- eIF-1 and eIF-3 bind the small ribosomal subunit
- ternary complex binds the small ribosomal subunit
- eIF-4 directs the complex towards the 5’ end of mRNA by binding the 5’ cap region (pre-Initiation complex)
What is the ternary complex?
Part of the first step of translation initiation and is the activated eIF-2 bound to met-tRNA
What is the pre-initation complex contain?
The ternary complex bound to the small ribosomal subunit and bound to the 5’ cap region of mRNA via eIF-4
What happens during Step 2 of translation initiation?
eIFs come off
Large ribosomal subunit binds the pre-initiation complex
Initiation complex formed
What are the 3 important sites of an assembled ribosome and what do they do?
P - peptidyl site - occupies by methionyl-tRNA
A- acceptor site - incoming aminoacyl tRNAs bind this site
E - ejection site - aids in removal of tRNAs
What are the 3 steps of elongation of translation?
- Aminoacyl-tRNAs bind at A site via eEF-1-GTP providing energy via GTP
- Formation of peptide bond between adjacent amino acids via peptidyltransferase activity of large ribosomal subunit
- Translocation of the peptidyl-tRNA to the P site and then the uncharged tRNA in the P site moves to the E site for ejection
What enzyme forms the peptide bond between two adjacent amino acids during translation?
Peptidyltransferase of the large ribosome subunit
What are the 3 steps of termination of translation?
- Stop codon enters the A site and a eukaryotic release factor (eRF) bound to GTP pairs with stop codon
- Newly synthesized peptide chain is released and GTP supplies the energy
- Ribosomal subunits dissociate from each other and mRNA and tRNA is released
What do polysomes do?
Read one mRNA molecule to synthesize multiple copies of one protein.
What is the mode of action of streptomycin?
mRNA is misread (no initiation complex)
What is the mode of action of tetracycline?
Inhibition of binding of amino-acyl tRNA to the A site. Can’t start translation
What is the mode of action of Cholramphenicol ?
Inhibition of peptidyl transferase activity - effects elongation and formation of peptide bond
What is the mode of action of erythromycin?
Inhibition of translocation of peptidyl-tRNA to the P site - effects elongation
What is the mode of action of neomycin/gentamycin?
Mistranslation of mRNA codon so incorrect aa-tRNAs are incorporated - effects initiation
Gray Baby Syndrome
Caused by adverse reaction to chloramphenicol
Mitochondrial ribosomes appear similar to procaryotic ribosomes so they get attacked.
Symptoms are ashen-gray skin, blue lips, and blue nail bed
What is synthesized by Corynebacterium diptheriae and what are its targets, mode of action, and symptoms?
Diphtheria toxin
Targets eEF2 - elongation factor
Inhibits eEF-2 by ADP ribosylation - insertion of bulky group
Symptoms are lesions in the upper respiratory tract causing necrotic injury to epithelial cells
What targets the 28S rRNA, causing conformational changes? What is its mode of action?
Ricin
Ricin is glycoprotein with N-glycosidase activity. Cleaves an adenine base from the 28S rRNA of the large ribosomal subunit.
Cleavage causes loss of binding of elongation factors to the large ribosomal subunit and turns protein synthesis off.
Extremely toxic
What are 3 Post-Translational Fates of proteins?
- Folding
- Post-translational modifications
- Transportation to the correct cellular component
Explain protein folding
Proteins are folded into 3D structures and the folding is assisted by chaperone proteins
What do chaperone proteins do?
Bind the hydrophobic region of partially folded peptides and guide them to correct folding. Found in cytoplasm and ER
What is an example of a heat shock protein (HSP)?
Hsp90 - major chaperone that repair damaged proteins due to heat or stress
What is the the process of carbohydrate addition to a protein and what proteins commonly have carb additions?
Glycosylation
Common glycosylated proteins include antibodies, protein hormones, growth factors, and cytokines. They move a lot so they need to be stable and easily solubilized
What enzymes assist in glycosylation and how do they work?
Glycosyltransferases
Form glycosidic bonds and each glycotransferase is specific for both the donor sugar and the acceptor molecule
Why is glycosylation of proteins important?
Increases solubility and stability
Important for formation of recognition sites
What are the two types of glycosylation?
N-linked glycosylation
O-linked glycosylation
Explain N-linked glycosylation
N-acetylglucosamine (sugar monomer) directly binds protein and is attached to the amide group of aspargine. More monomers can attach to the sugar to form an oligosaccharide. Process starts in ER and continues into Golgi
A phosphorylated lipid embedded in the ER that acts as a carrier of the oligosaccharide core
Dolichol phosphate
What is the first step in N-linked glycosylation? What occurs?
Building of a Universal Oligosaccharide
Synthesized by glycosyltransferases attached to cytosolic and luminal face of RER
Enzymes build oligosaccharide on dolichol phosphate first
First monomer is N-acetylglucosamine
More monomer are added until oligosaccharide is made
Transferred to nascent polypeptide chain
What is the second step in N-linked glycosylation? What occurs?
Specific Modifications
Made in Golgi apparatus and leads to 2 types of N-glycosylated proteins:
1. High mannose type
2. Complex type - containing sialic acid, fucose, N-acetyl glucosamine, galactose
What occurs during O-linked glycosylation?
Sugar monomer is added to hydroxyl group of serine or threonine
Other sugar are added
Occurs on fully folded proteins and takes place in the golgi
Explain ABO Blood types and their antigens
A, B, and O blood group antigens each consist of short oligosaccharide chain
A antigen - contains N-acetylgalactosamine added by GalNAc transferase
B antigen - contains one extra galactose added via Gal transferase
O antigen has nothing but short olig. chain
What does each blood type express?
A - A antigen
B - B antigen
AB - AB antigen
O - none
What proteins have lipid groups added to them and what are the examples?
Proteins that associate with membranes (membrane anchoring of proteins)
Palmitoylation - Palmitic acid added to SH of cysteine
Prenylation - Isoprenoid added to SH of cysteine
Myristoylation - Myristic acid added to NH of glycine
Addition of ADP-ribose to arginine or glutamine. What example did we see of this?
ADP-ribosylation. Diphtheria toxin
Phosphorylation
Addition of phosphate group on serine, threonine, or tyrosine. Serves to inactivate or activate proteins
Acetylation
Addition of acetyl group to lysine. Histone are modified by HATs which relax chromatin and allow gene expression
Proteolysis
Cleavage of peptide bonds to remodel proteins and activate them
Hydroxylation
Hydroxylation of prolyl and lysyl residues in collagen. Hydroxyproline is unique to collagen and important for proper formation of collagen fibrils.
Disorder in Collagen Processing and Copper Metabolism. What symptoms occur and what is affected?
Menkes Disease.
Mutations in ATPase of copper transporting alpha gene and lysine hydroxylase is dependent on copper so copper accumulates in small intestine and kidneys. Other areas like brain have copper deficiency.
Symptoms include depigmented hair and hypotonia
Where do proteins get targeted to typically?
- Peroxisome
- Lysosome
- Nucleus
- Mitochondria
Explain protein targeting to mitochondria
Mitochondrial protein synthesized in cytosol with N-terminal leader sequence (pre-sequence)
They require chaperones and then the complex of TOM and TIM (translocases of outer and inner mitochondrial membranes) provided channel for transportation
Matrix proteases cleave leader sequence
What proteins are subjected to degradation? And how are they degraded?
Regulatory proteins - rapid turnover
Damaged proteins
Degraded via lysosomes and proteasomes
What enzymes are in lysosomes?
Hydrolytic enzymes
What modification is critical for direction of enzymes to a lysosome?
Lysosomal enzymes are post translationally modified by glycosylation with mannose-6-phosphate
I-cell Disease
Transfer of a phosphate to mannose during post-translational modification of lysosomal enzymes is impaired.
Lysosomal enzymes cannot enter lysosomes and are secreted in serum so lysosomes are nonfunctional.
Clinical features include fibroblasts with inclusion bodies, coarse facial features, and bone fractures and slow motor abilities.
Explain proteasomal degradation
Target cytoplasmic proteins.
Proteins are marked with ubiquitins, which are activated by E1 ubiquitin activating enzyme.
Conjugated to polyubiquitins by E2 ubiquiting conjugating enzyme
Ligated to the target protien by E3 ligase and then polyubiquinated proteins are targeted for destruction via proteasome
Mutation in the gene encoding chloride channel membrane protein (CFTR)
Cystic Fibrosis.
Mutation to the gene causes disorder in N-linked glycosylation and misfolding of CFTR so CFTR is subjected to proteasomal degradation
Clinical features are stickiness of fluids.
Symptoms include cough, repeated lung infections, and damaged pancreas.
alpha1- antitrypsin deficiency
Alpha 1-antitrypsin is expressed in the liver.
Deficiency occurs from mutations in allelic variants (Z,S) of the gene encoding alpha1-antitrypsin protein
Mutations cause misfolding of alpha1-antitrypsin and accumulates of aggregates in ER damaging the liver cell
Symtpoms include liver cirrhosis and chronic pulmonary disease due to lack of alpha1-antritrypsin to fight infections
Do patient who express the M allele of the gene encoding alpha1-antitrypsin develop the deficeincy?
No, no disease!
