Protein synthesis

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  • Created by: Huzaima1
  • Created on: 30-12-18 23:32

Introduction

  • Cells synthesise large numbers of diverse proteins which all have different functions, e.g;
  • structural - such as assemble the plasma membrane, cytoskeleton and other organelles 
  • hormones, antibodies, contractile elements in muscular tissue, enzymes 
  • Proteins determine the physical and chemical characteristics of cells and therefore of the organisms formed from them 
  • Proteome - refers to all of an organism's proteins 
  • In the process called Gene Expression - a gene's DNA is used as a template for synthesis of a specific protein - through the processes transcription (the information encoded in a specific region of DNA is transcribed (copied) to produce a specific molecule of RNA) and translation (the RNA attaches to a ribosome where the information contained in RNA is translated into a corrosponding sequence of amino acids to form a new protein molecule) 
  • DNA and RNA - store genetic information as sets of three nucleotides - called a base triplet
  • Each base triplet - transcribed as a complementary sequence of three nucleotides - a codon 
  • A given codon specifies a particular amino acid 
  • The genetic code = a set of rules that relate the base triplet sequence of DNA to the corrosponding codons for RNA and the amino acids they specify
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Transcription - occurs in the nucleus;

  • the genetic information represented by the sequence of base triplets in DNA serves as a template for copying the information into a complementary sequence of codons 
  • RNA polymerase - catalyses the transcription of DNA 
  • Only one of the two DNA strands serves as a template for RNA synthesis 
  • Transcription begins at a special nucleotide sequence called a promoter - located near the beginning of a gene, where RNA polymerase attaches to the DNA 
  • During transcription, complementary bases pair - C,G,T and A in DNA pair up with G,C,A, and U in RNA (Uracil replaces Thymine in the RNA strand) 
  • Transcription of the DNA strand ends at another special nucleotide sequence called a terminator, which specifies the end of the gene - this is where RNA  polymerase detaches from the transcribed RNA strand and the DNA strand
  • Note: not all parts of the gene code for protein 
  • regions within a gene which do NOT code for proteins are called introns; regions which DO code for proteins are called exons 
  • the transcribed RNA (which contains both introns and exons) is called pre-mRNA 
  • Splicing occurs - a process which removes the introns using enzymes called small nuclear ribonucleoproteins which cut the introns and splice the exons together - resulting product = functional mRNA; exits through a nuclear envelope pore to reach the cytoplasm 
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Translation

  • occurs in the cytoplasm - carried out by ribosomes 
  • translation is where the nucleotide sequence in an mRNA molecule specifies the amino acid sequence of a protein 
  • the small subunit of a ribosome has a binding site for mRNA; the larger subunit has three binding sites for tRNA molecules; a P, A and E site 
  • AUG = start codon but also the codon for the amino acid methionine = always first aa in a growing polypeptide chain (tRNA anticodon = UAC)
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Lecture notes

Transcription - 

  • RNA polymerase breaks apart the DNA strands 
  • A single strand of mRNA is transcribed from the template strand using the base pair rule 
  • In mRNA, the nucleobase T is substituted by U 
  • Required nucleotides for mRNA synthesis are found freely in the nucleus

Pre mRNA capping - 

  • Pre mRNA needs a 5" cap
    • the cap is composed of phosphorylated 7-methyl guanosine which is added to the 5" end of the mRNA by guanyltransferase 
    • ensures mRNA is exported out of the nucleus 
    • blocks degradation of mRNA by 5' exonucleases 
    • promotes translation 
    • [Eukaryotic mRNA has a structure known as a cap at the 5'' end, a sequence of adenine nucleotides at the 3'' end, and a coding region in between containing codons that dictate the sequence of amino acids in a protein or relay a signal]

 

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Lecture notes

Polyadenylation of pre-mRNA 

  • Pre mRNA needs a 3' poly A-tail: 
  • the pre-mRNA is cleaved by an endonuclease near a signal AAUAAA sequence at the 3' end 
  • Approx 200 adenosine residues are then added at the cleavage site by poly-A-polymerase 
  • this poly-A-tail protects the mRNA from degradation by 3'' exonucleases 
  • the poly-A-tail also aids in termination of transcription, ensures export from the nucleus and is important in translation 

Alternative splicing - alternative splicing of a pre-mRNA sequence can produce different proteins from the same gene 

Summary - mRNA consists of: 

  • 5' cap 
  • 5' UTR [untranslated region] 
  • coding region [to be translated into a protein] (with a start and stop codon)
  • 3' UTR [untranslated region]
  • poly(A) tail 

Proteins consist of amino acids covalently linked with peptide bonds into a polypeptide chain 

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Lecture notes - genetic code and proteins

Basic and acidic aa: Hydrophobic residues (fold on inside) + Hydrophilic residues = important for protein folding 

The Genetic Code 

  • DNA base triplets represent each amino acid 
  • Base triplets in mRNA are called codons 
  • There are 20 amino acids and 64 (48 ) mRNA codons:
  • More than one codon codes for each of the 20 amino acids (the genetic code is degenerate)
  • First two bases in the codon are the most crucial: give tolerance against mutations , e.g: 
  • UCA,UCC,UCG,UCU - all code for Serine 
  • Some mRNA codons have special roles: 
  • START codon = AUG (methionine)
  • STOP codons = UAG, UAA,UGA
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Lecture notes - genetic code, proteins and mutatio

  • the order of bases in the genetic code in DNA codes for the appropriate sequence of amino acids in the proteins 
  • Transcription (DNA to mRNA) and translation (mRNA to protein) are important processes for protein synthesis 
  • Substitutions of bases changing the codon can change the corrosponding amino acid 
  • this can change protein structure significantly and may even result in the creation of stop codons in the wrong places 

Mutations in Sickle Cell Anaemia: 

  • single point mutation - more common in countries with high rates of malaria as it prevents contraction of the disease in the victim 
  • where glutamic acid in the normal amino acid sequence [ valine-histadine-leucine-threonine-proline-glutamic acid-glutamic acid] mutates to valine 
  • the change in amino acid sequence causes haemoglobin to crystallise when oxygen levels are low, causing the sickle shape which gets stuck in small blood vessels
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Lecture notes - clinical consequence of mutations

  • Accumulation of sickled cells in small blood vessels leads to;
    • downstream tissue ischaemia (not enough oxygen reaches the tissues) - causing pain and infarction
    • in severe cases, organ damage and ischaemic stroke 
  • inherited and chronic disease with periodic painful attacks
  • common in sub-saharan Africa where sickled red blood cells provide protection against the malarial parasite, thus giving individuals with the mutation a selective advantage 
  • in the US where malaria is not endemic, the sickle cell trait is slowly dissappearing from the african-american population
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Lecture notes - protein synthesis

  • DNA code is transcribed into mRNA in the nucleus 
  • Protein synthesis takes place outside the nucleus 
  • DNA is too big to leave the nucleus 
  • mRNA small and mobile 
    • mRNA leaves the nucleoplasm via nuclear pores in the nuclear envelope and enters the cytoplasm
    • mRNA can then travel to ribosomes for translation of the DNA code into proteins 
  • Ribosomes
  • are composed of ribosomal RNA and ribosomal proteins 
  • consist of a 60S and a smaller 40S subunit 
  • can be free or attached to the RER

tRNAs link mRNA with amino acid assembly - so they are assembled in the correct sequence

  • tRNA molecules have complementary base triplets that match up to the mRNA code 
  • there is a tRNA for each codon;tRNA molecules have amino acids attached

 

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Lecture notes - tRNA structure and translation

  • a tRNA bound to an amino acid (ester bond) = aminoacyl tRNA (also known as a charged tRNA) 
  • a tRNA molecule which has had its amino acid removed = a deacylated or uncharged tRNA
  • a tRNA molecule bound to growing polypeptide chain = peptidyl tRNA 

Translation 

  • during translation, the amino acid sequence is determined by the 3 base sequence making up the codon 
  • the process in the ribosome builds the polypeptide chains that will make up the protein
  • tRNA from the cytosol carries the amino acid and anticodon 
  • complementary base pairing occurs with the anticodon and codon 
  • two amino acids join using a peptide bond 
  • tRNA is released after the amino acid is removed 
  • THERE IS ALWAYS 3 TRNA BOUND AT ANY TIME
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Lecture notes - definitions

  • Initiation - binding of ribosomes to the 5' end of mRNA and hydrogen binding of the anticodon of an aminoactylated tRNA carrying methionine on the AUG start codon 
  • Elongation - the addition of further amino acids to the growing polypeptide chain brought by corrosponding aminoacylated tRNAs. Peptidyl transferase creates covalent peptide bonds between the amino acids 
  • Termination - when the stop codon (UAA,UAG,UGA) is reached and the peptide and ribosomal subunits are released 

Fate of synthesised polypeptide

  • has to acquire a secondary structure; alpha helices and beta pleated sheets 
  • has to fold into a tertiary structure
  • has to assemble into a quaternary structure if appropriate 
  • proteins have to reach their destinations 
    • proteins destined for use within the cytoplasm are synthesised on free ribosomes 
    • proteins destined for secretion out of the cell are synthesised on ribosomes attached to the rough ER (exocytosis - out of the cell) 
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Lecture notes - secreted proteins

  • synthesised on ribosomes attached to the RER 
    • RER is a system of flattened cavities 
    • lined by thin membrane running from nuclear envelope and into the cytoplasm and with many ribosomes on its surface 
    • provides compartment for protein synthesis 
  • secreted proteins have special signal sequences which interact with the RER membrane
  • these proteins are incorporated into vesicles (small spherical compartments made from RER membrane) for transport to the golgi apparatus 
  • vesicles move to the golgi complex/apparatus 
    • GA is a system of flattened plate-like cavities 
    • lined by a thin membrane 
    • post-translational modification of proteins occurs in the GA cavities. E.g. glycosylation of membrane spanning proteins 
  • the now modified proteins traverses the GA and is packaged into secretory vesicles 
  • protein containing secretory vesicles move to the cell membrane, fuse with it and expel their contents into the extracellular space (exocytosis) 
  • some specialised GA vesicles called lysosomes contain enzymes which can digest old organelles 
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