Translation of mRNA

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• Translation of mRNA-
• This is the biological polymerization of
  amino acids into polypeptide chains.
• Translation is the process by which the
  nucleotide sequence of mRNA is converveted to the
  amino acid sequence of a polypeptide.
• In the first step of the process, all the
  components needed for translation come together.
• These components include mRNA, tRNA and ribosomal
  units.

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• The mRNA transcript is a linear sequence of
  nucleotides carrying genetic information and it
  is single-stranded.
• Every three bases of mRNA (a triplet)
  specifies an amino acid to be added to a growing
  polypeptide chain the relationship between the
  triplets and the corresponding amino acids is the
  genetic code.
• each base triplet of mRNA is called a
  codon.
• the genetic code is nearly universal for
  all forms of life.

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• Genetic code- Genetic information stored in DNA
  and transferred to RNA during the process of
  transcription is present as three letter code
  words
• Characteristics of the genetic code
• (a) Each group of 3 ribonucleotides is called as
  the codon and specifies one amino acid the code
  is thus a triplet.
• (b) The code is unambiguous, meaning each triplet
  specifies only a single amino acid.
• ( c ) The code contains start and stop
  signals, these initiate and terminates
  translation
• (d) Once translation of mRNA begins, the codons
  are read one after the other with no breaks
  between them.
• (e) A code of 4 nucleotides taken three at a time
  could provide 43 number of combinations- clearly
  more than needed to code all the amino acids
  (there are only 20 A. As)

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• Amino acids are the building blocks (monomers) of
  proteins. 20 different amino acids are used to
  synthesize proteins. The shape and other
  properties of each protein is dictated by the
  precise sequence of amino acids in it. Each
  amino acid consists of an alpha carbon atom to
  which is attached
• a hydrogen atom
• an amino group (hence "amino" acid)
• a carboxyl group (-COOH). This gives up a proton
  and is thus an acid (hence amino "acid")
• one of 20 different "R" groups. It is the
  structure of the R group that determines which of
  the 20 it is and its special properties. The
  amino acid shown here is Alanine.

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• Degeneracy and Wobble hypothesis
• The genetic code is degenerate almost all amino
  acids are specified by 2,3 or 4 different codons.
  Ex. Serine is coded by UCU, UCC, UCA, UCG, AGU
  and AGC
• In a set of codons specifying the same amino
  acid, the first two letters are same, with only
  the third differing. Crick postulated that the
  initial 2 ribonucleotides of triplet codes are
  more critical than the third member in attracting
  the correct tRNA. This is known as the wobble
  hypothesis. This hypothesis allows the anticodon
  of a single form of tRNA to pair with more than
  one triplet in mRNA. Therefore for the 64 triplet
  codons, a minimum of about 30 different tRNA
  species is only required.
• tRNAs 10-15 of the total RNA of a cell. Has
  about 75-90 nucleotides.
• Mature tRNA has a clover leaf structure (2ry
  structure)

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• tRNA molecule

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• It fuctions as an interpreter between nucleic
  acid and peptide sequences by picking up amino
  acids and matching them to the proper codons in
  mRNA.
• There are two important locations on a tRNA
  molecule that help it do this
• At the bottom of the loop are three
  ribonucleotides grouped together in an anticodon.
  
• An anticodon is complementary to an mRNA codon.
• An anticodon can recognize and bind to its
  complementary mRNA codon.
• Some tRNAs can recognize more than one codon
  because there is a relaxation of the
  complementation rule of base pairing between the
  anticodon and codon in the third position.
• This relaxation is called the Wobble Hypothesis.
• 2. At the 3 end of the tRNA strand is where the
  amino acid attaches to the tRNA molecule.

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• Each tRNA carries one amino acid that corresponds
  to an mRNA codon,
• The proper amino acid is joined to the tRNA by
  the enzyme aminoacyl-tRNA synthetase.
• There is one type of this enzyme for each amino
  acid and the active site of each fits only the
  specific combination of the proper amino acid and
  tRNA.
• In tRNA the bases of the anticodon are modified,
  tRNA has Inosinic acid and similar derivatives
  which could form hydrogen bonds with U, C or A
• Charging of tRNA
• Before translation can proceed, the tRNA
  molecules must be chemically linked to their
  respective amino acids. This process called
  charging occurs under the direction of enzymes
  called aminoacyl tRNA synthetases.

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• Fig. 13.5
• Aminoacyl tRNA synthetases are highly specific
  enzymes as they recognize only one amino acid
  so 20 synthetases are specific to each amino
  acid.
• Ribosomes - A ribosome is made of rRNA and
  proteins.
• A ribosome is composed of two subunits, a large
  subunit and a small subunit. Both sub units
  together is called as a monosome.
• These subunits join to form a functional ribosome
  when they attach to mRNA.
• There are differences between prokaryotic and
  eukaryotic ribosomes. Fig 13-1

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• Translation the process
• Initiation - In the first step in protein
  synthesis, the small 30S subunit of the ribosome
  binds to the mRNA molecule (Diagram 1) this
  contains triplet codon (AUG,) at which protein
  synthesis starts. A set of initiation factors
  (proteins) enhances the binding affinity.The
  charged formylmethionyl tRNA then binds to the
  mRNA codon in P site of the small subunit of the
  ribosome. This aggregate is the initiation
  complex. Then the large subunit binds to the
  complex.
• Fig 13.6

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• In bacteria, the first AA-tRNA to initiate
  translation is always a formyl derivative of
  methionine called FMet-tRNA In bacteria this
  binding involves a sequence up to 6
  ribonucleotides AGGAGG which precedes the initial
  AUG start codon of mRNA. This sequence is known
  as Shine-Dalgarno sequence
• In eukaryotes, synthesis is started by a special
  initiation Met-tRNA, but the methionine is not
  formylated. However, the initial methionine is
  usually split off from the finished polypeptide
• A G G A G G A U G

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• Elongation- Increase of the growing polypeptide
  chain by one amino acid is called elongation. The
  sequence of the second triplet in mRNA dictates
  which charged tRNA molecule will become
  positioned at the A site. Once it is present,
  peptidyl transferase catalyzes the formation of
  the peptide bond, which links the 2 amino acids.
  Fig. 13-7

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• A molecule of water is released ( it is a
  condensation reaction) This only happens after
  hydrolysis of a GTP into GDP which allows the
  elongation factor to leave. This delay allows for
  proof reading as a wrong tRNA would leave before
  the reaction takes place.

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• The role of the small subunit during elongation
  is one of decoding the triplets present in mRNA
  and the large subunit is the place where peptide
  bonds are synthesized.

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• Termination-
• Termination of the polypeptide occurs when the
  ribosome reaches a "Stop" Codon.
• Chain termination leads to the release of a
  polypeptide, and tRNA, and the dissociation of
  the ribosome into 30S and 50S subunits. Stop
  codons are triplets which are not recognized by
  any tRNA (UAA, UAG, UGA), the two proteins the
  GTP-dependant releasing factors cleave the
  polypeptide chain from the terminal tRNA
  releasing it from the translation complex. This
  is initiated when R1, R2 recognizes the stop
  codons. R1 recognizes UAG and recognizes UAA and
  UGA).
• The polypeptide released will be processed in
  different parts of the cell, depending on its
  role, and destination. All the processing
  involved depends on the polypeptide sequence,
  therefore on the mRNA sequence (and therefore on
  the original DNA base sequence).

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• If a termination codon should appear in the
  middle of an mRNA molecule as a result of
  mutation the same process occurs and the
  polypeptide chain is prematurely terminated.
• Differences between Prokaryotic and eukaryotic
  translation
• In eukaryotes translation occurs in ribosomes
  which are larger and whose rRNA and protein
  components are more complex.
• Eukaryotic mRNA are longer lived.
• The RNA processing step is absent in prokaryotes
  and capping is essential for efficient
  translation. But kozak sequence (5 ACCAUG)
  similar to Shine Dalgarno sequence is present in
  Eukaryotes around the start codon (AUG)
  functions in initiating translation

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• One gene one enzyme hypothesis
• Biochemical reactions are controlled by enzymes
  and often are organized into chains of reactions
  known as metabolic pathways. Loss of activity in
  a single enzyme can inactivate an entire pathway.
• Archibald Garrod, in 1902, proposed the
  relationship through his study of alkaptonuria-
  large quantities "alkapton (homogenistic acid)
  in urine in affected individuals Garrod
  suspected a blockage of the pathway to break this
  chemical down, and proposed that condition as "an
  inborn error of metabolism". He also discovered
  alkaptonuria was inherited as a recessive
  Mendelian trait

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• George Beadle and Edward Tatum in 1930s
  established the connection Garrod suspected
  between genes and metabolism. They used X rays to
  cause mutations in strains of Neurospora. These
  mutations affected single genes and single
  enzymes in specific metabolic pathways. Beadle
  and Tatum proposed the "one gene one enzyme
  hypothesis" Fig. 13-11
• the chemical reactions occurring in the body are
  mediated by enzymes, and since enzymes are
  proteins and thus heritable traits, there must be
  a relationship between the gene and proteins.
• George Beadle, proposed that mutant eye colors in
  Drosophila was caused by a change in one protein
  in a biosynthetic pathway.

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• Analysis of biochemical pathways
• Phenylketonuria-This inherited human metabolic
  disorder results when the phenylalanine and
  tyrosine metabolic pathway is blocked.
• Phenylalanine Phenylpyruvic acid
  elevated
• 
  levels can cause
• 
  mental retardation in
  Phenylalanine hydroxylase new
  borns
• Phenylketonuria block
• Tyrosine
• Alkaptonuria block Homogenistic acid oxidase
• Homogenistic acid
  Maleylacetoacetic acid
  
• block
• Fig 13-10

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• One gene one polypeptide- One gene one enzyme
  hypothesis could although explain the blockage of
  many biochemical pathways, it was not convincing
  for some since they couldnt imagine how a mutant
  enzyme could bring variations in the phenotypes.
• However two factors
• Nearly all enzymes are proteins but all proteins
  are not enzymes
• All proteins are specified by information stored
  in genes
• Changed the one gene one enzyme hypothesis to one
  gene one polypeptide.
• These modifications became apparent during the
  analysis of hemoglobin structure in individuals
  afflicted with sickle cell anemia.
• This was the first direct evidence that genes
  specify proteins other than enzymes

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• Sickle-cell anemia (h) is a recessive allele in
  which a defective hemoglobin is made, causing
  pain and death to those individuals homozygous
  recessive for the trait. Normal and affected
  individuals result from the homozygous genotypes
  HbAHbA and HbsHbs. Heterozygotes make both normal
  and "sickle cell" hemoglobins and are carriers of
  the defective gene. Linus Pauling made the first
  findings that the hemoglobin isolated from
  diseased and normal individuals differ in their
  rates of electophoretic mobility. So there should
  be a chemical change in the the normal and
  diseased types. Later Vernon Ingram discovered
  that the normal and sickle-cell hemoglobins
  differ by only 1 (out of a total of 300) amino
  acid. Valine was substituted for glutamic acid at
  the 6th position of the ß chain accounting for
  the peptide difference.
• Human hemoglobin contains two identical a chains
  of 141 amino acids and two identical ß chains of
  146 amino acids in its quarternary structure

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• The above results led to the confirmation of
• Single gene provides inform. For a single
  polypeptide
• The concept for inherited molecular diseases were
  confirmed.