GEN 272 Introductory Molecular Genetics

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GEN 272Introductory Molecular Genetics

• Lecture Eight

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Central Dogma of Molecular Biology
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Translation requires ribosomes and tRNAs

• Translation refers to the biological
  polymerization of amino acids into polypeptide
  chains
• The process occurs in association with ribosomes,
  which serve as the nonspecific workbenches
• Also involved during translation are intermediate
  molecules (i.e. transfer RNAs or tRNAs), which
  adapt specific triplet codons in mRNA to their
  correct amino acids
• a specific tRNA molecule contains within its
  nucleotide sequence three consecutive
  ribonucleotides complementary to the codon,
  called the anticodon, which can base-pair with
  the codon
• another region of this tRNA molecule is
  covalently bonded to its corresponding amino acid
• Inside the ribosome, H-bonding of tRNAs to mRNA
  holds the amino acid in proximity so that a
  peptide bond (between amino acids) can be formed
• the process occurs continuously until the entire
  mRNA runs through the ribosome, and amino acids
  are polymerized into a polypeptide

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Ribosomal Structure
Fig. 14-1
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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rRNA Components

• In E. coli, the genome contains seven copies of a
  single sequence that encodes all three components
  23S, 16S, and 5S
• the initial transcript of these genes produces a
  30S RNA molecule that is enzymatically cleaved
  into these smaller components
• coupling of the genetic information encoding
  these three rRNA components ensures that,
  following multiple transcription events, equal
  quantities of all three will be present as
  ribosomes are assembled
• In eukaryotes, many more copies of a sequence
  encoding the 28S, 18S, and 5.8S components are
  present
• for example, in Drosophila (120 copies/haploid
  genome are transcribed into a molecule of 34S)
  in Xenopus laevis (gt 500 copies/haploid genome)
• in mammalian cells the initial transcript is 45S
  and the rRNA genes (referred to as rDNA), are
  part of a moderately repetitive DNA fraction and
  are present in clusters at various chromosomal
  sites
• each cluster consists of tandem repeats, with
  each unit separated by a noncoding spacer DNA
  sequence

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tRNA Structure

• Very small in size (only 75 90 nucleotides) and
  stable
• Display a nearly identical structure in bacteria
  and eukaryotes
• in both organisms the tRNAs are transcribed as
  larger precursors, which are cleaved into 4S
  mature tRNA molecules
• for example, in E. coli, tRNAtyr is composed of
  77 nucleotides, but its precursor contains 126
  nucleotides
• In 1965, Robert Holley and colleagues reported on
  the complete sequence of tRNAala isolated from
  yeast
• interestingly, they identified a number of
  nucleotides that are unique to tRNAs
• these include inosinic acid (Inosine) and others
  containing modified nitrogenous bases than
  expected in G, C, A, and U (see Figure 14-2)
• these modified structures (sometimes referred to
  as unusual, rare, or odd bases) are created
  following transcription and thus, further
  illustrate the concept of posttranscriptional
  modification
• in such cases, the unmodified bases are inserted
  during transcription and, subsequently, enzymatic
  reactions catalyze the chemical modifications to
  the base

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Cloverleaf Model of tRNA

• Holleys analysis of the tRNAala led him to
  propose the 2D cloverleaf model of tRNA
• the model was based on previous knowledge
  that
  tRNAs demonstrated secondary structure
  due to
  (internal) base pairing
• this base pairing creates a series of paired
  
  stems and unpaired loops resembling the shape
  of
  a cloverleaf
• interestingly, loops consistently contained
  
  modified bases that did not generally form
  
  base pairs
• Alanine is specified by GCU, GCC, and GCA
• an anticodon sequence complementary to one of
  
  these codons in the tRNAala molecule was
  found
  in the form of CGI (3 to 5)
  in one of the loops
  of the cloverleaf (Note I
  can pair with U, C,
  and A)
• thus, the anticodon was established

Variable loop
Dihydrouracil
Pseudouracil
Fig. 14-3
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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Other features of tRNA molecules

• Investigation of other tRNA species revealed that
  
• at the 3end, all tRNAs contain the sequence
  pCCA-3, which are added post-transcriptionally
  (note an amino acid is covalently joined to the
  terminal A residue )
• all tRNAs contain 5-G at the other
  
  end of the molecule
• the lengths of various stems and loops
  
  are very similar
• each tRNA (examined thus far) contains
  
  an anticodon complementary to the
  
  known amino acid codon for which it is
  
  specific
• all anticodon loops are present in the
  
  same position of the cloverleaf
• Note pseudouracil and dihydrouracil bases
  
  are required for proper
  folding and operation
  of the tRNA.
  Also, the T?C-loop and the
  
  D-loop are needed for binding to the
  
  ribosome and for recognizing the enzyme
  which sticks the amino acids onto the tRNA

Fig. 14-4
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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Amino Acid Structure

• There are 20 amino acids that serve as the
  building blocks of proteins (see Fig. 14-16)
• Each amino acid has the following groups
• a carboxyl (COOH) group
• an amino (NH2) group
• a radical (R) group (also called a side chain)
  that is bound covalently to a central carbon atom
• The R group gives each amino acid its
  chemical
  identity (20 R groups divided into
  four main classes)

Fig. 14-16
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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R Group (Nonpolar Hydrophobic)
Fig. 14-16
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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R Group (Polar Hydrophilic)
Fig. 14-16
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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R Group (Polar Positively charged, basic)
Fig. 14-16
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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R Group (Polar Negatively charged, acidic)
Fig. 14-16
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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Peptide Bond
HOH water
H2N---AA1---AA2---AA3---AA4---AAn-2---AAn-1---AAn-
--COOH (Polypeptide)
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Charging tRNA molecules

• Before translation can proceed, tRNA molecules
  must be chemically linked to their respective
  amino acids
• This activation process, termed charging (or
  aminoacylation), occurs under the direction of
  enzymes called aminoacyl tRNA synthetases
• In theory, because of the Wobble hypothesis,
  there are at least 32 different tRNAs and only 20
  synthetases (one for each amino acid)
• The charging process involves
• converting an amino acid into an activated form
  via a reaction with ATP to create an
  aminoacyladenylic acid. A covalent linkage is
  formed between the 5-phosphate group of ATP and
  the carboxyl end of the amino acid. This molecule
  remains associated with the synthetase enzyme,
  forming a complex that reacts with a specific
  tRNA molecule
• the amino acid is transferred to the appropriate
  tRNA molecule and bonded covalently to the A
  residue at the 3 end
• Aminoacyl tRNA synthetases are highly specific
  because they recognize only one amino acid and
  only the tRNAs corresponding to that amino acid,
  called isoaccepting tRNAs

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Charging tRNA molecules
Fig. 14-5
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Klug et al. (2006). Concepts of Genetics, 8th
Edition
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Charging tRNA molecules
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Clark and Russell (2005) Molecular Biology made
simple and fun, 3rd Ed.
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Translation Initiation Components

• When not involved in translation, ribosomes are
  dissociated into their large and small subunits
• Translation initiation (in E. coli) involves the
  small ribosomal subunit, an mRNA molecule, a
  specific charged initiator tRNA molecule, GTP,
  Mg2, and a number of proteinaceous initiation
  factors (IFs, see Table 14.1)
• IFs are initially part of the small subunit and
  required to enhance the binding affinity of the
  various translational components
• unlike ribosomal proteins, IFs are released from
  the ribosome once initiation is completed

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Translation Initiation Components
Fig. 14-5
Klug et al. (2006). Concepts of Genetics, 8th
Edition
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Take Home Message

• Translation is the polymerization of amino acids
  of amino acids into a polypeptide chain
• There are 20 amino acids with each contain a
  basic structure including an amino group, a
  carboxyl group, and a radical group (side chain)
  covalently bonded to a central carbon atom
• The process requires ribosomes, tRNA molecules,
  amino acids, a number of proteinaceous molecules
  and an energy providing group
• The bond occurring between two amino acids is
  called a peptide bond. A chain of several amino
  acids adjacently bonded to each other is termed a
  polypeptide

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