From DNA to Protein

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Chapter 7

• From DNA to Protein

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DNA to Protein

• DNA acts as a manager in the process of making
  proteins
• DNA is the template or starting sequence that is
  copied into RNA that is then used to make the
  protein

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Central Dogma

• One gene one protein

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Central Dogma

• This is the same for bacteria to humans
• DNA is the genetic instruction or gene
• DNA ? RNA is called Transcription
• RNA chain is called a transcript
• RNA ? Protein is called Translation

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Expression of Genes

• Some genes are transcribed in large quantities
  because we need large amount of this protein
• Some genes are transcribed in small quantities
  because we need only a small amount of this
  protein

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Transcription

• Copy the gene of interest into RNA which is made
  up of nucleotides linked by phosphodiester bonds
  like DNA
• RNA differs from DNA
• Ribose is the sugar rather than deoxyribose
  ribonucleotides
• U instead of T A, G and C the same
• Single stranded
• Can fold into a variety of shapes that allows RNA
  to have structural and catalytic functions

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RNA Differences
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RNA Differences
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Transcription

• Similarities to DNA replication
• Open and unwind a portion of the DNA
• 1 strand of the DNA acts as a template
• Complementary base-pairing with DNA
• Differences
• RNA strand does not stay paired with DNA
• DNA re-coils and RNA is single stranded
• RNA is shorter than DNA
• RNA is several 1000 bp or shorter whereas DNA is
  250 million bp long

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Template to Transcripts

• The RNA transcript is identical to the
  NON-template strand with the exception of the Ts
  becoming Us

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RNA Polymerase

• Catalyzes the formation of the phosphodiester
  bonds between the nucleotides (sugar to
  phosphate)
• Uncoils the DNA, adds the nucleotide one at a
  time in the 5 to 3 fashion
• Uses the energy trapped in the nucleotides
  themselves to form the new bonds

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RNA Elongation

• Reads template 3 to 5
• Adds nucleotides 5 to 3 (5 phosphate to 3
  hydroxyl)
• Synthesis is the same as the leading strand of DNA

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RNA Polymerase

• RNA is released so we can make many copies of the
  gene, usually before the first one is done
• Can have multiple RNA polymerase molecules on a
  gene at a time

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Differences in DNA and RNA Polymerases

• RNA polymerase adds ribonucleotides not
  deoxynucleotides
• RNA polymerase does not have the ability to
  proofread what they transcribe
• RNA polymerase can work without a primer
• RNA will have an error 1 in every 10,000
  nucleotides (DNA is 1 in 10,000,000 nucleotides)

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Types of RNA

• messenger RNA (mRNA) codes for proteins
• ribosomal RNA (rRNA) forms the core of the
  ribosomes, machinery for making proteins
• transfer RNA (tRNA) carries the amino acid for
  the growing protein chain

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DNA Transcription in Bacteria

• RNA polymerase must know where the start of a
  gene is in order to copy it
• RNA polymerase has weak interactions with the DNA
  unless it encounters a promoter
• A promoter is a specific sequence of nucleotides
  that indicate the start site for RNA synthesis

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RNA Synthesis

• RNA pol opens the DNA double helix and creates
  the template
• RNA pol moves nt by nt, unwinds the DNA as it
  goes
• Will stop when it encounters a STOP codon, RNA
  pol leaves, releasing the RNA strand

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Sigma (?) Factor

• Part of the bacterial RNA polymerase that helps
  it recognize the promoter
• Released after about 10 nucleotides of RNA are
  linked together
• Rejoins with a released RNA polymerase to look
  for a new promoter

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Start and Stop Sequences
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DNA Transcribed

• The strand of DNA transcribed is dependent on
  which strand the promoter is on
• Once RNA polymerase is bound to promoter, no
  option but to transcribe the appropriate DNA
  strand
• Genes may be adjacent to one another or on
  opposite strands

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Eukaryotic Transcription

• Transcription occurs in the nucleus in
  eukaryotes, nucleoid in bacteria
• Translation occurs on ribosomes in the cytoplasm
• mRNA is transported out of nucleus through the
  nuclear pores

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RNA Processing

• Eukaryotic cells process the RNA in the nucleus
  before it is moved to the cytoplasm for protein
  synthesis
• The RNA that is the direct copy of the DNA is the
  primary transcript
• 2 methods used to process primary transcripts to
  increase the stability of mRNA being exported to
  the cytoplasm
• RNA capping
• Polyadenylation

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RNA Processing

• RNA capping happens at the 5 end of the RNA,
  usually adds a methylgaunosine shortly after RNA
  polymerase makes the 5 end of the primary
  transcript
• Polyadenylation modifies the 3 end of the
  primary transcript by the addition of a string of
  As

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Coding and Non-coding Sequences

• In bacteria, the RNA made is translated to a
  protein
• In eukaryotic cells, the primary transcript is
  made of coding sequences called exons and
  non-coding sequences called introns
• It is the exons that make up the mRNA that gets
  translated to a protein

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RNA Splicing

• Responsible for the removal of the introns to
  create the mRNA
• Introns contain sequences that act as cues for
  their removal
• Carried out by small nuclear riboprotein
  particles (snRNPs)

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snRNPs

• snRNPs come together and cut out the intron and
  rejoin the ends of the RNA
• Intron is removed as a lariat loop of RNA like
  a cowboy rope

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Benefits of Splicing

• Allows for genetic recombination
• Link exons from different genes together to
  create a new mRNA
• Also allows for 1 primary transcript to encode
  for multiple proteins by rearrangement of the
  exons

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Summary
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RNA to Protein

• Translation is the process of turning mRNA into
  protein
• Translate from one language (mRNA nucleotides)
  to a second language (amino acids)
• Genetic code nucleotide sequence that is
  translated to amino acids of the protein

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Degenerate DNA Code

• Nucleotides read 3 at a time meaning that there
  are 64 combinations for a codon (set of 3
  nucleotides)
• Only 20 amino acids
• More than 1 codon per AA degenerate code with
  the exception of Met and Trp (least abundant AAs
  in proteins)

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Reading Frames

• Translation can occur in 1 of 3 possible reading
  frames, dependent on where decoding starts in the
  mRNA

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Transfer RNA Molecules

• Translation requires an adaptor molecule that
  recognizes the codon on mRNA and at a distant
  site carries the appropriate amino acid
• Intra-strand base pairing allows for this
  characteristic shape
• Anticodon is opposite from where the amino acid
  is attached

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Wobble Base Pairing

• Due to degenerate code for amino acids some tRNA
  can recognize several codons because the 3rd spot
  can wobble or be mismatched
• Allows for there only being 31 tRNA for the 61
  codons

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Attachment of AA to tRNA

• Aminoacyl-tRNA synthase is the enzyme responsible
  for linking the amino acid to the tRNA
• A specific enzyme for each amino acid and not for
  the tRNA

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2 Adaptors Translate Genetic Code to Protein
? 2
?1
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Ribosomes

• Complex machinery that controls protein synthesis
• 2 subunits
• 1 large catalyzes the peptide bond formation
• 1 small binds mRNA and tRNA
• Contains protein and RNA
• rRNA central to the catalytic activity
• Folded structure is highly conserved
• Protein has less homology and may not be as
  important

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Ribosome Structures

• May be free in cytoplasm or attached to the ER
• Subunits made in the nucleus in the nucleolus and
  transported to the cytoplasm

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Ribosomal Subunits

• 1 large subunit catalyzes the formation of the
  peptide bond
• 1 small subunit matches the tRNA to the mRNA
• Moves along the mRNA adding amino acids to
  growing protein chain

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Ribosomal Movement
E-site

• 4 binding sites
• mRNA binding site
• Peptidyl-tRNA binding site (P-site)
• Holds tRNA attached to growing end of the peptide
• Aminoacyl-tRNA binding site (A-site)
• Holds the incoming AA
• Exit site (E-site)

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3 Step Elongation Phase

• Elongation is a cycle of events
• Step 1 aminoacyl-tRNA comes into empty A-site
  next to the occupied P-site pairs with the codon
• Step 2 C end of peptide chain uncouples from
  tRNA in P-site and links to AA in A-site
• Peptidyl transferase responsible for bond
  formation
• Each AA added carries the energy for the addition
  of the next AA
• Step 3 peptidyl-tRNA moves to the P-site
  requires hydrolysis of GTP
• tRNA released back to the cytoplasmic pool

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Initiation Process

• Determines whether mRNA is synthesized and sets
  the reading frame that is used to make the
  protein
• Initiation process brings the ribosomal subunits
  together at the site where the peptide should
  begin
• Initiator tRNA brings in Met
• Initiator tRNA is different than the tRNA that
  adds other Met

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Ribosomal Assembly Initiation Phase

• Initiation factors (IFs) catalyze the steps not
  well defined
• Step 1 small ribosomal subunit with the IF
  finds the start codon AUG
• Moves 5 to 3 on mRNA
• Initiator tRNA brings in the 1st AA which is
  always Met and then can bind the mRNA
• Step 2 IF leaves and then large subunit can
  bind protein synthesis continues
• Met is at the start of every protein until
  post-translational modification takes place

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Eukaryotic vs Procaryotic

• Procaryotic
• No CAP have specific ribosome binding site
  upstream of AUG
• Polycistronic multiple proteins from same mRNA
• Eucaryotic
• Monocistronic one polypeptide per mRNA

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Protein Release

• Protein released when a STOP codon is encountered
• UAG, UAA, UGA (must know these sequences!)
• Cytoplasmic release factors bind to the stop
  codon that gets to the A-site alters the
  peptidyl transferase and adds H2O instead of an
  AA
• Protein released and the ribosome breaks into the
  2 subunits to move on to another mRNA

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Polyribosomes

• As the ribosome moves down the mRNA, it allows
  for the addition of another ribosome and the
  start of another protein
• Each mRNA has multiple ribosomes attached,
  polyribosome or polysome

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Regulation of Protein Synthesis

• Lifespan of proteins vary, need method to remove
  old or damaged proteins
• Enzymes that degrade proteins are called
  proteases process is called proteolysis
• In the cytosol there are large complexes of
  proteolytic enzymes that remove damaged proteins
• Ubiquitin, small protein, is added as a tag for
  disposal of protein

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Protein Synthesis

• Protein synthesis takes the most energy input of
  all the biosynthetic pathways
• 4 high-energy bonds required for each AA addition
• 2 in charging the tRNA (adding AA)
• 2 in ribosomal activities (step 1 and step 3 of
  elongation phase)

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Summary
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Ribozyme

• A RNA molecule can fold due to its single
  stranded nature and in folding can cause the
  cleavage of other RNA molecules
• A RNA molecule that functions like an enzyme
  hence ribozyme name