Protein Synthesis Flashcards
RNA Polymerase functions
Does not require free 3’ OH end
Catalyses formation of phosphodiester bonds between adjacent RNA ribonucleotides
Recognises template at promoter
Binds to promoter → unwinds DNA double helix to expose nucleotide bases
Transcription (prokaryotes)
- Initiation
Sigma factor (part of RNA polym) recognises promoter
RNA polymerase binds to promoter
Unwinding of DNA double helix
Sigma factor released after addition of 10 mRNA nucleotides → allow RNA polymerase to continue down DNA template strand - Elongation
Addition free ribonucleotide triphosphate groups (ATP,CTP,GTP,UTP) to the template strand via CBP
Synthesis of growing strand in 5’ → 3’ (ie move along template strand in 3’ - 5’)
Formation of phosphodiester bonds between ribonucleotides catalysed by RNA polymerase
Energy for phosphodiester bond formation comes from removal of 2P from incoming nucleotides
Transcription bubble formed → DNA unwound ahead of bubble, synthesised RNA peels away from template strand, DNA rewinds - Termination
Termination sequence is transcribed by RNA polymerase
This termination sequence is complementary to part of the RNA sequence → binds upon itself → forms hairpin loop → RNA dissociates from RNA polymerase and template DNA
Transcription (eukaryotes)
- Initiation
General transcription factors recognise and bind to TATA box on promoter
GTF recruit RNA polymerase and ensure correct positioning
Formation of Transcription Initiation Complex → unwinding of DNA double helix - Elongation
Synthesis of growing strand In 5’ → 3’ (moves along template in 3’ to 5’)
Addition of free ribonucleotide triphosphates to template strand nucleotides via CBP
RNA polymerase catalyses formation of phophodiester bonds between adjacent ribonucleotides
Energy for bond formation comes from removal of 2P from incoming ribonucleotides
Transcription bubble → DNA is unwound ahead of bubble, RNA peels off from template strand, DNA rewinds - Termination
Termination sequence transcribed by RNA polymerase
Polyadenylation signal (AAUAAA)
Certain proteins recruited to cleave mRNA strand downstream of polyadenylation signal → halt further transcription
Pre-mRNA → post transcriptional modifications
Post transcriptional modifications
5’ capping
3’ polyadenylation
Splicing
5’ capping
Addition of 7-methylguanosine residue to 5’ end of pre-mRNA
Catalysed by mRNA guanyltransferase
5’ cap joined to pre mRNA in reverse orientation
5’ - 5’ triphosphate bridge formed
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Forms barrier to protect mRNA from degradation by 5’ exonucleases (hydrolytic enzymes) in cytoplasm
Facilitates export of mature mRNA from nucleus to cytoplasm
Helps mRNA bind to ribosome for translation
3’ polyadenylation
Addition of poly A tail to 3’ end of pre mRNA
Catalysed by poly-(A)-polyadenylase → catalyses formation of phosphodiester bonds between adjacent adenosine residues
Added after cleaving of mRNA downstream of polyadenylation signal
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Stabilises mRNA as template for translation
→ survive longer in cytoplasm = more proteins translated
Protects from degradation by 3’ exonucleases (hydrolytic enzymes)
Aids export of mature mRNA from nucleus to cytoplasm
No poly A tail = no translation
Splicing
Removal of introns
In nucleus, requires energy in form of ATP
Splice sites are varied, GU- -AG
Several snRPs recognise and bind to splice sites via CBP, complex together (formation of phosphodiester bonds) to form spliceosome
SnRP=protein x snRNA
Spliceosome = specific 3d configuration where exons are brought together, lariat formation
Introns excised, exons brought together = mature mRNA
Mature mRNA leaves nucleus for translation in cytoplasm
Alternative splicing
All introns removed
Constitutive exons remain
Alternative exons may or may not be excised out
Differences in exons present may affect folding, function, of polypeptide
Allows for one gene to code for many different proteins = ↓ amt of genes required to be carried in genome
Genetic code
Relationship between nucleotide sequence of mRNA and amino acid sequence of polypeptide it codes for
Degenerate but not ambiguous
More than one codon can code for the same amino acid
One codon can only code for one amino acid
Non-overlapping
Translation starts at AUG, travels down in 5’-3’ sequence, one codon at a time
Triplet code
4 nucleotide bases, 3 mRNA = 1 codon
4x4x4 possible codons → 20 amino acids
AUG → start codon, methionine
UGG, UAG, UAA → stop codons
Universal
All codons code for the same amino acids across all organisms
Translation preparation
Amino-acyl tRNA is formed
Catalysed by aminoacyl tRNA synthetase (ATS)
20 ATS specific for 20 amino acids → specificity due to R group
tRNA anticodon binds to specific ATS via CBP between anticodon and active site of ATS
Addition of amino acid to 3’ end of tRNA → covalent linkage
Allows for amino acids to form peptide bond with carboxyl end of the growing polypeptide chain
Energy in form of ATP hydrolysis required
Ribosomes
Small ribosomal subunit : contains mRNA binding site (rRNA is complementary to mRNA)
30S p
40S e
Large ribosomal subunit : contains 3 tRNA binding sites - Aminaocyl tRNA, Peptidyl tRNA, Exit site
50S p
60S e
P: 70S
E: 80S
Ribosome formation
Genes coding for rRNA found in nucleolus, transcripted to form rRNA, rRNA remains in nucleolus
Genes coding for ribosomal proteins found in nucleus, undergoes transcription, modifications, exported out of nucleus via nuclear pores into cytoplasm for translation
Transported back into nucleus from complexing with rRNA to form ribosomes
Exported out of cell into cytoplasm → free ribosomes or attached to rER
Translation (prokaryotes)
- Preparation
- Initiation
MRNA binding site on small ribosomal subunit (30S) binds to Shine Dalgarno sequence downstream of 5’ end
Initiator tRNA carrying formyl-methionine binds to AUG via CBP (anticodon) → future P site
Large ribosomal subunit binds → energy needed from GTP hydrolysis
Translation initiation complex formed - Elongation
Aminoacyl tRNA binding
Corresponding aminoacyl tRNA binds to empty A site via CBP
Peptide bond formation
Peptidyl transferase (ribozyme, part of large ribosomal subunit) catalyses peptide bond formation between adjacent amino acids → growing polypeptide chain
TRNA at P site loses its amino acid
Translocation
Translation initiation complex moves down mRNA in 5’ to 3’ direction down 3 nucleotide bases
Requires energy in form of GTP hydrolysis
Aminoacyl tRNA carrying growing polypeptide chain initially at A site is now at P site → A site empty
tRNA at P site is now empty tRNA at E site → released
Process continues, corresponding aminoacyl tRNA binds, more amino acids added to C end
- Termination
Complex eventually reaches termination sequence (UGG UAG UAA)
Release factor (protein) binds to A site → addition of H2O ∴ hydrolyse bond between polypeptide chain and tRNA → release polypeptide chain
Translation initiation complex, ribosomal subunits etc dissociates
Translation (eukaryotes)
- Preparation
- Initiation
Initiator tRNA carrying methionine binds to small ribosomal subunit
Initiation factors bind to small ribosomal subunit with initiator tRNA to stabilise structure
MRNA binding site (contains rRNA) on small ribosomal subunit x initiator tRNA binds to 5’ Untranslated Region (UTR) upstream of AUG
Small ribosomal subunit x initiator tRNA moves down mRNA in 5’ to 3’ direction until it reaches AUG, where anticodon of initiator tRNA binds to start codon via CBP → P site
Large ribosomal subunit binds → energy in form of GTP hydrolysis and dissociation of initiation factor
Formation of translation initiation complex - Elongation
Aminoacyl tRNA binding
Corresponding tRNA binds via CBP of anticodon to the empty site
Peptide bond formation
Peptidyl transferase (ribozyme, part of large ribosomal subunit) catalyses formation of peptide bonds between adjacent amino acids → growing polypeptide chain
P site tRNA loses its amino acid
Translocation
Translation initiation complex moves down mRNA in 5’ to 3’ direction down 3 nucleotide bases → energy from GTP hydrolysis
Aminoacyl tRNA carrying growing polypeptide chain is now at P site, A site empty
Free tRNA at E site, leaves
Corresponding aminoacyl tRNA binds to A site via CBP between anticodon and mRNA
Process continues, amino acids continuously added to carboxyl end of growing polypeptide chain
- Termination
Reaches stop codon (UGG UAG UAA)
Release factor (protein) is accepted into A site → addition of H2O to hydrolyse bond between polypeptide chain and tRNA → release of polypeptide chain
Dissociation of translation initiation complex
Protein folding
Protein is folded into specific 3D conformation
Aided by chaperone proteins
Synthesised by:
Free ribosomes- folding occurs spontaneously, protein generally remains in cytoplasm
RER ribosomes- must enter cisternae to be folded into specific shape
Signal peptide at N terminus of polypeptide chain allows for polypeptide to be transported into cisternae via channel proteins
Signal peptide is cleaved by enzymes after entry into the cisternae
These proteins tend to be secreted out of the cell, inserted into cell membrane, transported into lysosome
