Protein Synthesis

  • Protein Synthesis
    • Follows central dogma


DNA > RNA > Protein


DNA codes for proteins
RNA

    • Ribonucleic acid
    • Single-stranded with ribose sugar and uracil (replaces thymine)
    • G - C | A _U


  • Transcription
    • DNA to pre-mRNA
    • Occurs in the nucleus
  • RNA Processing
    • Pre-mRNA to RNA
  • Translation
    • mRNA to protein
    • Occurs in the cytoplasm, with ribosomes at the rough ER
    • Creates 20 essential amino acids


  • Ribosomes are found on the rough endoplasmic reticulum



    From DNA to Protein
    • DNA has to be transcribed to RNA One DNA strand serves as a template
    • RNA is first formed as pre-RNA and has to undergo processing before it become mRNA
      • Processing requires 2 steps
    • mRNA then leaves the nucleus and enters the cytoplasm
    • RNA is translated to produce proteins, a complex multi-step process utilizing ribosomes


  • Expression of Genes from DNA
    • DNA sequence predicts the mRNA sequence which determine how the protein is produced
    • On DNA sequence are units called genes that determine the exact protein to be produced
    • Proteins are made of amino acids which the DNA sequence determines which amino acids are produced through the genetic code
    • These genes eventually determine the biochemical and phenotypic traits in an organism
    • There are 20,000 genes in humans


Gene

DNA

Sequence of DNA/RNA that codes for a molecule that has a function. The transmission of genes to organisms offspring is the basis of the inheritance of phenotypic traits

A molecule composed of 2 chains which coil around each other to form a double helix carrying the genetic instruction used in the growth, development and functioning of organisms.





  • A gene contains a promoter region and combination of intros and exons

    Exons
    • Expressed sequences
    • Coding regions
      • Translated into amino acid sequences plus the leader and trailer
  • Introns (need to get rid of)
    • Intervening sequences
    • Non coding segments


  • Transcription
    • Template DNA strand 3' to 5'
    • mRNA pairs with DNA
    • 3 pairs of nucleotides in mRNA is a codon
    • TATA Box
      • A eukaryotic promoter, how we identify gene




  • Transcription Pt2
    • Process involves "reading" the DNA strand to produce a complementary mRNA strand
    • At the extreme 5' end of the DNA molecule is a promoter sequence that contains a TATA sequence
    • The enzyme RNA polymerase 2 together with transcription factors bind to the promoter region to form a transcription initiation complex
    • The RNA polymerase adds nucleotides at a rate of 40 per second to begin a new mRNA strand in the 5' to 3' direction
    • The transcription ends when the polymerase reaches the polyadenylation signal (AAUAAA)


  • RNA polymerase 2
    • Take the DNA and make it into RNA
    • Goes 5' to 3'


  • RNA splicing
    • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides


  • Spliceosomes, Collection of Enzymes
    • Remove introns and joins exons together


  • Eukaryotic cells modify RNA after transcription
    • Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm
    • At the 5' end of the pre-mRNA molecule a modified form of guanine is added the 5' cap


  • 5'caps
    • Helps to protect mRNA from hydrolytic enzymes
    • Functions as an "attach here" signal for ribosomes
  • Poly-A tail
    • Helps transport out of the nucleus and provides greater protection to the mRNA
    • Inhibits mRNA degradation


  • Completed RNA processing in the Nucleus
    • The 5' G-P-P-P regions protects mRNA from being degraded by enzymes in the cytoplasm. Site to signal ribosomes to attach
    • The Poly-A tail inhibits mRNA degradation and facilitates export out of the nucleus
    • DNA gene sequence that is 27,000 long may produce an mRNA molecule is only 1,200 nucleotides long


  • RNA processing
    • Involves inverting pre-mRNA into RNA that can leave the nucleus and enter the cytoplasm where it can be transcribe
    • 3 steps
      • Add 5' region
      • Add Poly-AAA tail
      • Splice out introns
    • Splicing is removing introns and merging exons together to form mature mRNA
    • mRNA can leave nucleus now


  • Translation (Produce polypeptide/protein)
    • Mature mRNA leave nucleus into cytoplasm
    • mRNA must be read according to Dictionary of the Genetic Code. Specifies which amino acids will form the new protein
    • Read in groups of 3 nucleotides called codon. Each codon specifies an amino acid
    • There are 20 amino acids
    • Triplet code is universal
    • It is redundant
      • The same amino acid can be coded by different codons
    • Code is unambiguous
      • One codon will never give rise to more than one amino acid


  • Start Codon - AUG

     Stop Codon - UAA, UAG, UGA it codes for water molecule to break bond



    Function of Ribosomes
    • Made up of small and large sub-units
    • Contain ribosomal RNA (rRNA)
    • Since ribosomes are very abundant in the cell, rRNA is the most abundant type of nucleic acid in the cell
    • A Site
      • Aminoacyl-tRNA binding site
    • P Site
      • Peptidyl-tRNA binding site
    • E site
      • Exit site


  • Translation Step 1 - Initiation
    • Small sub-unit of ribosomes binds to the 5' cap region of mRNA
    • An initiator transfer RNA (t-RNA) arrives at the P-site (peptidyl-tRNA binding site) carrying a methionine amino acid which is encoded by the "start" codon. Energy is required (GTP)
    • On the t-RNA molecuels are "anti-codon" sites which correspond to each codon on the mRNA
    • For each 20 amino acids, there are 20 different t-RNA molecules that correspond to the codons
    • Large subunit of the ribosomes binds to the small subunit to form the translation initiation complex


  • Anti-codon
    • Pairs with codon nitrogenous base pairings


  • T-RNAs
    • Each molecule has an amino acid attachment site at one end and an anti-codon site at the other end
    • Each time a codon is read from the mRNA molecule a corresponding t-RNA molecules arrives, carrying the corresponding amino acid as specified by the Dictionary of the genetic code
    • t-RNA carrying the amino acid arrives at the A site on te ribosome
    • Attachment of the amino acid to the correct t-RNA is achieved by the action of an enzyme called Aminoacyl-tRNA synthetase


  • Translation Step 2 - Elongation
    • Once initiation step is started, the mRNA is read though the ribosome complex as codons which specify the next amino acids
    • Corresponding t-RNA carrying the amino acid arrives at the A-site
    • The amino acid is transferred from the t-RNA sitting at the P-Site to the A-site and a bond peptide bond is formed with the existing amino acids. Results in a polypeptide forming each time a new amino acid is added
    • The t-RNA in the P site transfers to the E-site. The tRNA in the A-site moves to the P-site to make room for the new t-RNA. Cycle continues


  • Translation Step 3 - Termination
    • At the point of termination of the translation, as top codon (UAA, UAG, UGA) is reached
    • Codes for a release factor which is a water molecule
    • Causes the polypeptide chain to be released and the last t-RNA molecule leaves the ribosome complex
    • Small and large subunits separate
    • Formation of a polypeptide (protein molecule) is an energy requiring process (GTP)
    • Takes 1 minute for a cell to produce a new polypeptide




  • Mutation Examples

     Silent mutation
    • Changes 1 letter
    • Still codons for the same amino acid
    • Rat > RaT
    • GGC > GGU both codes for Gly
  • Nonsense no protein
    • Deletes or adds 1 letter
    • Shifts nitrogenous pairings down by 1
    • She saw > shs aw_
    • She Ssa w___

Normal

Mutation

DNA

TAC TTC

ATG AAG

TAC ATC

ATG TAG

mRNA

AUG AAG

AUG UAG

Amino Acid

Met Lys

Met Stop codon





  • Mis-sense different Protein
    • Delete or Add 3 letters
    • She saw big red dog
      • She saw big ___ dog
      • She saw big old dog



    • Point mutation also mis-sense mutation
      • Single base pair change occurs
      • T turns to C
      • Results in a single nucleotide polymorphism (different) called SNP

Normal

Point Mutation

DNA

CTT

GAA

CAT

GTA

mRNA

GAA

GUA

Hemoglobin

Glu

Val




  • Mutations
    • Change in the DNA sequence that results in a change in the mRNA sequence
    • May or may not result in a change in the amino acid produce and protein produced
    • Point mutation
      • Single base pair change occurs
      • T turns to C
      • Results in a single nucleotide polymorphism (different) called SNP
    • May have results during DNA replication where an incorrect base was added and the polymerase enzyme did not detect it. Or could be from X-rays or UV light


  • Sickle cell trait and malaria resistance
    • Carriers of the sickle cell allele (Ss) are more resistant to malaria
    • Malaria is a serious disease caused by parasites, spready by bites from infected mosquitoes
    • Enter red bloods cells, causes fever and vomiting and can be fatal
    • It is common where malaria is widespread as their genes mutated to become more resistant to malaria, that mutation so happens to be the sickle cell allele
    • 1 single mutation in 1 baby led to the disease of sickle cell anemia 7,300 years ago


  • How the S gene affects malaria parasite
    • Heterozygotes do to malaria
    • Tend to have lower numbers of parasitized red cells in blood
    • Decreased incidence of the two forms of severe life0threatening malaria
      • Cerebral malaria and malaria with severe anemia
      • Rarely die of malaria


  • Parkinson's Associated Gene
    • 5 potential areas on a gene where mutation can occur
    • Number 2 is the most prevalent mutation in the Parkinson population


  • Importance of mutations
    • Many mutations may have no effects, many result in devasting disease in humans
      • Parkinson's, Alzheimer's, cancer etc
    • During evolution, mutations can have beneficial roles by increasing genetic diversity.
      • Mutation that reduced keratin production resulted in humans having less body hair compaired to chimpanzees