Unit 11: Protein synthesis/translation Flashcards
Describe the genetic code
- triplet code- each codon is 3 nucleotides long
- non-overlapping- codes do not overlap
- commaless or no punctation- codes are not spaced apart
- (almost) universal - almost all organisms use this
- degenerate- most amino acids can be coded for by more than one codon (aka synonyms)
- unambiguous- each codon can code for only one amino acid
what is the start codon
AUG= Met
what is the direction of peptide synthesis
5’ to 3’
What is the open reading Frame (ORF)
- the sequence from start to stop codon
- the start frame is determined by the where the start codon (AUG) is
what does aminoacyl-tRNA synthetase do
- it is the enzyme that connects an amino acid with a corresponding tRNA
- uses ATP. converts to AMP and PPi.
- activates AA and charges tRNA. AA is considered activated when connected to tRNA and tRNA is considered charged when connected to an AA
- produces aminoacyl-tRNA
where is the amino acid attached on the tRNA?
it is attached to the 3’-OH of CCA end of the tRNA or acceptor arm
describe the shine-dalgarno sequence
- is purine rich
- found in prokaryotes translation start
- base-pairs with rRNA small subunit.
- helps orient the ribosome to align with start codon
describe the prokaryotic start signal vs. eukaryotic start signal
prokaryotic:
shine dalgarno sequences (this sequence base pairs with rRNA small subunit) followed by start codon AUG that codes for fMet
eukaryotic:
first AUG from 5’ end codes for H2N-Met
describe the polypeptide synthesis mechanism
synthesized from n-term to c-term.
- new AA is added to carboxyl terminus by peptide transferase function of ribosome
- amino acids react in activated form as aminoacyl-tRNA
- mRNA translated 5’ to 3’
- this allows the concurrent transcription and translation in prokaryotes
what are the stages of protein synthesis
- Activation of AA- activate carboxyl group of AA to allow formation of peptide bond linkage to tRNA. produces aminoacyl-tRNA
- Initiation
- elongation
- termination
- protein folding and post translational processing
where does activation of AA take place
cytoplasm
where does initiation, elongation, and termination take place
the ribosomes (found in the cytoplasm and ER)
where does posttranslational processing take place
cytoplasm, ER, and Golgi
What bonds anticodon to codon?
non-covalent interactions
what enzyme forms the high energy bond between tRNA and AA? what type of bond is it
aminoacyl-tRNA syntheses. there is one synthetase per amino acid and recognizes all tRNAs for that specific AA.
- most have proofreading capacity. only proofreading done in translation
- it is a thioester bond; a covalent bond. this stores the energy that makes peptide bonds possible
ways of altering mRNA sequence
point mutations- single base pair changes: giving silent mutation, missense or nonsense mutation
frameshift mutations: insertion or deletion of nucleotides within coding sequence leading to altered reading frame
what is a missense mutation vs. a nonsense mutation
their is a base pair change that results in a different amino acid
nonsense mutation is a base pair change that results in an early stop codon
how many codons are there
- but only 61 actually code for amino acids.
3 are stop codons (UAG, UAA, UGA)
What is the wobble hypothesis
1st and 2 codon position in codon and anticodon follow strict Watson-crick base pairing
3rd mRNA codon and 1st tRNA anticodon are flexible
- reduces the number of tRNAs needed
tRNA structure
- secondary and tertiary are similar for all tRNAs
- many modified bases
- 50% of all bases are paired
- stem-loop structures don’t pair and create structural diversity.
- AA binds to the 3’ CCA on the acceptor stem
- 5’ end usually G rich
describe the thermodynamics of the aminoacyl-tRNA reaction
- aminoacyl-tRNA synthetase requires ATP to produce the ester bond.
- in the process 2 high energy bonds are consumed for AA activation.
- AMP + PPi are produced
- rxn is exergonic
describe the aminoacyl-tRNA synthetase proofreading activity
- a separate editing site.
- only incorrectly bound amino acids fit into the site and the bond is hydrolyzed
- it is used by most syntheses but not all need them
what are isoaccepting tRNAs
tRNAs that recognize >1 codon for a specific AA
describe the main structure and function of a ribosome
- contains a small and large subunit.
- 70s prokaryotes (50S and 30s)
- 80s eukaryotes (40s and 60s)
- made of protein and RNA. is a ribonucleoprotein complex
- main catalytic function, peptide transferase activity, is performed by RNA specifically large subunit
- can be found free in cytoplasm or bound to membranes ( such as the RER in eukaryotes)
describe rRNA structure
- it is specific 3D structure with extensive intra-chain base pairing
- shape of rRNAs is highly conserved
what does the small subunit do (40s)
- binds mRNA and aminoacyl-tRNAs
- locates AUG start codon on mRNA
what does the large subunit do (60s)
binds to the small subunit after the start codon is located
has peptidyltransferase activity
what is expressosome formation
- prokaryotic translation
- complex of RNAP, mRNA and ribosome
what is the function of polysomes
- increase efficiency in prokaryotic translation
- are multiple ribosomes translating the same mRNA
what do initiation factors do? explain how initiation works in prokaryotes
they are required to bind the mRNA and beginning methionyl-tRNA to the small ribosomal subunit (eukaryotes)
- in prokaryotes the 30s subunit binds IF1 and IF3 then the mRNA
- then IF2-GTP binds the 30s and recruits fmet=tRNAfmet , which base pairs with the start codon
- the 50s subunit associates, IF2 hydrolyzes GTP, and IF1, IF2 and IF3 dissociate
Steps of elongation in prokaryotes
- binding of aminoacyl-tRNA (second tRNA) at A site
- GTP-EF-tu is attached. once proper base pairing is complete GDP-EF-Tu is released - peptide bond formation. peptide attached to tRNA in A site
- translocation of the ribosome
- EF-G (translocase) binds to A site, displaces tRNA with peptide into P-site, uncharged to E site
- GTP hydrolysis (loss of Phosphate) induces conformational change and release of EF-G
- mRNA shifted by one codon toward 3’ end
- uncharged tRNA dissociates and acceptor site can accept a new charged tRNA
describe termination in prokaryotes
- signaled by a stop codon
- no tRNA recognizing stop codons
- stop codons are bound by specialized release factors (RF-1, RF-2)
what do release factors in prokaryotes do?
- facilitate hydrolysis of ester linkage and peptide release
- release of uncharged tRNA in P-site
- Dissociate ribosome
eukaryotic translation vs. prokaryotic translation
- eukaryotic ribosomes are larger than prokaryotic ribosomes
- prokaryotes use no shine-dalgarno sequence, eukaryotes use Kodak sequence (ACCAUGG) includes most upstream AUG sequence after 5’
- eukaryotes have more initiation factors than prokaryotes
- special initiator tRNA does not use fmet. and met may be removed after
- circularized mRNA
-similar thermodynamics and reaction mechanism in elongation as in prokaryotes
elongation in eukaryotes?
- EF1-GTP brings in next codons (EF-Tu homolog)
- EF1beta/gamma regenerate EF1-GTP (EF-T homolog)
- EF2-GTP responsible for hydrolysis of phosphate for ribosome translocation (EF-G homolog)
describe termination in eukaryotes
- release factor eRF1 binds at A site
- this recognizes all stop codons and initiates hydrolysis of bound GTP and peptide-tRNA cleavage
- separation of mRNA and ribosome subunits done by ABCE1. requires ATP
how are ribosomal subunits recycled in eukaryotes?
by eIFs (eukaryotic initiation factors)
what does over expression of initiation factors in cancer cells do?
leads to gene amplification in human cancers
how is the process of translation exploited clinically?
- pharmaceuticals target prokaryotic translation inhibitors
- Use of antimicrobial therapy with prokaryotic translation inhibitors
- this exploits structural differences in eukaryotic and prokaryotic ribosomes.
how many ATPs are required for activation
2
how many ATPs are required for initiation
1
how many ATPs are required for elongation (each step)
2
* remember to account for the AA already in the active site
how many ATPs are required for termination
1
