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Chapter 7. From DNA to Protein. 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. Central Dogma. One gene – one protein. Central Dogma.

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chapter 7

Chapter 7

From DNA to Protein

dna to protein
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
central dogma
Central Dogma
  • One gene – one protein
central dogma1
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 atranscript
  • RNA  Protein is called Translation
expression of genes
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
transcription
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
transcription1
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
template to transcripts
Template to Transcripts
  • The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s
rna polymerase
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
rna elongation
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
rna polymerase1
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
differences in dna and rna polymerases
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)
types of rna
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
dna transcription in bacteria
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
rna synthesis
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
sigma factor
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
dna transcribed
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
eukaryotic transcription
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
rna processing
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
rna processing1
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 A’s
coding and non coding sequences
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
rna splicing
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)
snrnps
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
benefits of splicing
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
rna to protein
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
degenerate dna code
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)
reading frames
Reading Frames
  • Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA
transfer rna molecules
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
wobble base pairing
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
attachment of aa to trna
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
ribosomes
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
ribosome structures
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
ribosomal subunits
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
ribosomal movement
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)
3 step elongation phase
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
initiation process
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
ribosomal assembly initiation phase
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
eukaryotic vs procaryotic
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
protein release
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
polyribosomes
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
regulation of protein synthesis
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
protein synthesis
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)
ribozyme
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
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