Chapter 7
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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

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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


Rna differences

RNA Differences


Rna differences1

RNA Differences


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


Start and stop sequences

Start and Stop Sequences


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


Summary

Summary


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


2 adaptors translate genetic code to protein

2 ‘Adaptors’ Translate Genetic Code to Protein

 2

1


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)


Summary1

Summary


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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