Genes: Structure Replication and Expression

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Chapter 12

• Genes Structure Replication and Expression

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• Replication
• During mitotic division information is duplicated
  by DNA replication and is passed on to next
  generation
• daughter cells has exavcyt replica of the parent
  DNA

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Role of DNA in Protein synthesis

• DNA and protein synthesis involves
• Transcription- yields a ribonucleic acid (RNA)
  copy of specific genes
• Translation- uses information in messenger RNA
  (mRNA) to synthesize a polypeptide.
• Protein synthesis is assisted by RNA (tRNA) and
  ribosomal RNA (rRNA)

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Nucleic Acids
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Nucleic Acid StructureDeoxyribonucleic Acid (DNA)

• polymer of nucleotides
• contains the bases adenine, guanine, cytosine and
  thymine
• sugar is deoxyribose
• molecule is usually double stranded

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• DNA is a double-stranded molecule twisted into a
  helix (think of a spiral staircase).
• Each spiraling strand, comprised of a
  sugar-phosphate backbone and attached bases, is
  connected to a complementary strand by
  non-covalent hydrogen bonding between paired
  bases.
• The bases are adenine (A), thymine (T), cytosine
  (C) and guanine (G). A and T are connected by
  two hydrogen bonds. G and C are connected by
  three hydrogen bonds.

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DNA Structure Two Complementary Strands

• base pairing
• Adenine (purine) and thymine (pyrimidine) pair by
  2 hydrogen bonds
• Guanine (purine) and cytosine (pyrimidine) pair
  by 3 hydrogen bonds
• major and minor grooves form when the 2 strands
  twist around each other

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Nucleic Acid StructureRibonucleic Acid (RNA)

• polymer of nucleotides
• contains the bases adenine, guanine, cytosine and
  uracil
• sugar is ribose
• most RNA molecules are single stranded

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RNA Structure

• three different types which differ from each
  other in function and in structure
• messenger RNA (mRNA)
• ribosomal RNA (rRNA)
• transfer RNA(tRNA)

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The Organization of DNA in Cells

• In most bacteria DNA is a circular, double helix
• further twisting results in supercoiled DNA
• in bacteria the DNA is associated with basic
  proteins
• help organize the DNA into a coiled chromatin
  like structure

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• DNA Replication

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DNA Replication

• complex process involving numerous proteins which
  help ensure accuracy
• the 2 strands separate, each serving as a
  template for synthesis of a complementary strand
• synthesis is semi-conservative each daughter
  cell obtains one old and one new strand

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DNA Replication

• bidirectional from a single origin of replication

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DNA replication (arrows) occurs in both
directions from the origin of replication in the
circular DNA found in most bacteria.
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Rolling Circle Replication

• some small circular genomes (e.g., viruses and
  plasmids)
• replicated by rolling-circle replication
• Animation illustrating DNA replication by
  complementary base pairing

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Genes
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Gene Structure

• Gene
• the basic unit of genetic information
• also defined as the nucleic acid sequence that
  codes for a polypeptide, tRNA or rRNA
• linear sequence of nucleotides
• codons are found in mRNA and code for single
  amino acids
• reading frame
• organization of codons such that they can be read
  to give rise to a gene product

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Importance of Reading Frame
Figure 12.16
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Genes that Code for Proteins

• template strand directs RNA synthesis
• promoter is located at the start of the gene
• is the recognition/binding site for RNA
  polymerase
• functions to orient polymerase
• leader sequence is transcribed into mRNA but is
  not translated into amino acids
• Shine-Delgarno sequence important for initiation
  of translation

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Genes that Code for Proteins

• The Coding Region
• begins with the DNA sequence from 3-TAC-5
• produces codon AUG which codes for
  N-formylmethionine, a modified amino acid used to
  initiate protein synthesis in bacteria ( check
  fig.)
• coding region ends with a stop codon, immediately
  followed by the trailer sequence which contains a
  terminator sequence used to stop transcription

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Bacterial Gene Structure
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Genes That Code for tRNA and rRNA

• tRNA/rRNA genes have promoter (recognition/bindin
  g site for RNA polymerase), leader (is
  transcribed into mRNA), coding region, spacer and
  trailer regions (contains a terminator sequence
  used to stop transcription)
• during maturation process.

leader, spacer, and trailer removed during
maturation process
Figure 12.19a
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rRNA genes have promoter, leader, coding, spacer,
and trailer regions
spacer and trailer regions may encode tRNA
molecules
Figure 12.19b
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m
Fig. 12.20
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Transcription
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Transcription

• RNA is synthesized under the direction of DNA
• RNA produced has complementary sequence to the
  template DNA
• three types of RNA are produced
• mRNA carries the message for protein synthesis
• tRNA carries amino acids during protein synthesis
• rRNA molecules are components of ribosomes

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Transcription in Bacteria

• Definitions to understand protein synthesis
• in most bacterial RNA polymerases
• Holoenzyme can begin transcriptiongt What is
  Holoenzyme??
• the core enzyme is composed of 5 chains and
  catalyzes RNA synthesis
• the sigma factor has no catalytic activity but
  helps the core enzyme recognize the start of
  genes
• holoenzyme core enzyme sigma factor
• only the holoenzyme can begin transcription

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Transcription in Bacteria.

• Transcription in Bacteria is catalyzed by a
  single RNA polymerase.
• a reaction similar to that catalyzed by DNA
  polymerase for DNA syntehsis.
• ATP,GTP,CTP and UTP are used to produce a
  complementary RNA copy of the template DNA
  sequence

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http//www.vidoemo.com/yvideo.php?iM2FWVDJEcWuRpV
GJ0QTgreplication-transcription-and-translation
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Transcription Process
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Transcription Initiation

• Promoter
• site where RNA polymerase binds to initiate
  transcription is not transcribed

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Transcription Elongation

• after binding, RNA polymerase unwinds the DNA
• transcription bubble produced
• moves with the polymerase as it transcribes mRNA
  from template strand
• within the bubble a temporary RNADNA hybrid is
  formed

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Coupled Transcription and Translation in
Prokaryotes
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Proteins
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The Genetic Code

• mRNA sequence is translated into amino acid
  sequence of polypeptide chain (process
  translation).
• an understanding of the genetic code is necessary
  before translation is studied.

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Organization of the Code

• code degeneracy
• up to six different codons can code for a single
  amino acid
• sense codons
• the 61 codons that specify amino acids
• stop (nonsense) codons
• the three codons used as translation termination
  signals
• do not encode amino acids

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Translation
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Translation

• Translation of mRNA into protein
• synthesis of polypeptide is directed by sequence
  of nucleotides in mRNA
• Ribosome
• 70S ribosomes 30S 50S subunit
• site of translation
• polyribosome (polysome) complex of mRNA with
  several ribosomes

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• Translation of mRNA into protein
• Three phases
• Initiation
• Elongation
• Termination

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• During translation, the mRNA is "read" according
  to the genetic code which relates the DNA
  sequence to the amino acid sequence in proteins
• Each group of three base pairs in mRNA
  constitutes a codon, and each codon specifies a
  particular amino acid (hence, it is a triplet
  code).
• The mRNA sequence is thus used as a template to
  assemblein orderthe chain of amino acids that
  form a protein.

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Transfer RNA (tRNA) and Amino Acid Activation

• The tRNA molecules are adaptor moleculesthey
  have one end that can read the triplet code in
  the mRNA through complementary base-pairing, and
  another end that attaches to a specific amino
  acid
• attachment of amino acid to tRNA is catalyzed by
  aminoacyl-tRNA synthetases

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• The translation of mRNA begins with the formation
  of a complex on the mRNA (Fig. below).
• First, three initiation factor proteins (known
  as IF1, IF2, and IF3) bind to the small subunit
  of the ribosome.
• This preinitiation complex and a
  methionine-carrying tRNA then bind to the mRNA,
  near the AUG start codon, forming the initiation
  complex.

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The Ribosome
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• Methionine (Met) is the first amino acid
  incorporated into any new protein, however, it is
  not always the first amino acid in translation of
  protein.
• In many proteins, methionine is removed after
  translation.

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• The large ribosomal subunit binds to this
  complex, which causes the release of IFs
  (initiation factors) once the initiation complex
  is formed on the mRNA

• The large subunit of the ribosome has three sites
  at which tRNA molecules can bind
• The A (amino acid) site is the location at which
  the aminoacyl-tRNA anticodon base pairs up with
  the mRNA codon, ensuring that correct amino acid
  is added to the growing polypeptide chain.

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• The P (polypeptide) site is the location at which
  the amino acid is transferred from its tRNA to
  the growing polypeptide chain.
• Finally, the E (exit) site is the location at
  which the "empty" tRNA sits before being released
  back into the cytoplasm to bind another amino
  acid and repeat the process.

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• The initiator methionine tRNA is the only
  aminoacyl-tRNA that can bind in the P site of the
  ribosome, and the A site is aligned with the
  second mRNA codon.
• The ribosome is thus ready to bind the second
  aminoacyl-tRNA at the A site, which will be
  joined to the initiator methionine by the first
  peptide bond.

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Elongation of the Polypeptide Chain

• The next phase in translation is known as the
  elongation phase .
• Elongation cycle is the sequential addition of
  amino acids to growing polypeptide consists of
  three phases
• aminoacyl-tRNA binding
• transpeptidation reaction
• Translocation
• The above process need several Elongation factors
  ( EF)

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Elongation First, the ribosome moves along
the mRNA in the 5'-to-3'direction, which requires
the elongation factor G, in a process called
translocation
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..Elongation Cycle

• The tRNA which corresponds to the second codon
  can then bind to the A site, a step that requires
  elongation factors (in E. coli, these are called
  EF-Tu and EF-Ts) and GTP (guanosine triphosphate
  ) as an energy source for this acitivity.
• Upon binding of the tRNA-amino acid complex in
  the A site, GTP is cleaved to form guanosine
  diphosphate (GDP), then released along with EF-Tu
  to be recycled by EF-Ts for the next round.

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• .Elongation
• In the next step, peptide bonds between the
  now-adjacent first and second amino acids are
  formed through a peptidyl transferase activity.

• After the peptide bond is formed, the ribosome
  shifts, or translocates, again, thus causing the
  tRNA to occupy the E site.

• The tRNA is then released to the cytoplasm to
  pick up another amino acid.
• The A site is now empty and ready to receive the
  tRNA for the next codon.

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• .Elongation
• This process is repeated until all the codons in
  the mRNA have been read by tRNA molecules
• the amino acids attached to the tRNAs have been
  linked together in the growing polypeptide chain
  in the appropriate order.
• At this point, translation must be terminated,
  and the nascent protein must be released from the
  mRNA and ribosome.

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Final Phase in Elongation Cycle - Translocation

• Three simultaneous events
• peptidyl-tRNA moves from A site to P site
• ribosome moves down one codon
• empty tRNA leaves P site

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• Termination of Translation/protein synthesis
• Three termination codons ( Non-sense or stop
  codon) that are employed at the end of a
  protein-coding sequence in mRNA UAA, UAG, and
  UGA
• No tRNAs recognize these codons.
• Instead, release factors (RFs) helps in
  recognition of stop codons.
• Release factors are protein which binds and
  facilitates release of the mRNA from the ribosome
  and subsequent dissociation of the ribosome.

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Several ribosome can align on one mRNA strand
and forms several polypeptide chains each with 20
or more amino acids.

• http//www.vidoemo.com/yvideo.php?ibmNqSWlEcWuRpN
  TFoUWsdna-translation-animation

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Prokaryotic and Eukaryotic Translation

• The translation process is very similar in
  prokaryotes and eukaryotes.
• Although different elongation, initiation, and
  termination factors are used, the genetic code is
  generally identical.
• In bacteria, transcription and translation take
  place simultaneously, and mRNAs are relatively
  short-lived.

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• In eukaryotes, mRNAs have highly variable
  half-lives,
• are subject to modifications, and must exit the
  nucleus to be translated.

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References

• http//student.ccbcmd.edu/biotutorials/dna/fg12.ht
  mlhttp//www.accessexcellence.org/RC/VL/GG/dna_mol
  ecule.php
• http//www.nature.com/scitable/topicpage/Reading-t
  he-Genetic-Code-1042
• http//www.nature.com/scitable/topicpage/Translati
  on-DNA-to-mRNA-to-Protein-393