Protein Synthesis Chapter 17

Protein synthesis  DNA  Responsible for hereditary information  DNA divided into genes  Gene:  Sequence of nucleotides  Determines amino acid sequence in proteins  Genes provide information to make proteins

Protein synthesis DNA RNA protein

Central Dogma  Mechanism of reading & expressing genes  Information passes from the genes (DNA) to an RNA copy  Directs sequence of amino acids to make proteins

Protein synthesis  Transcription:  DNA sequence is copied into an RNA  Translation:  Information from the RNA is turned into an amino acid sequence

RNA  RNA (ribonucleic acid)  Single strand  Sugar –ribose (-OH on 2’ carbon)  Uracil instead of thymine

RNA  mRNA:  Messenger RNA  Transcribes information from DNA  Codons  (3 nucleotides) CGU  mRNA  Codes for amino acids  rRNA:  Ribosomal RNA  Polypeptides are assembled

RNA  tRNA:  Transfer RNA  Transports aa to build proteins  Positions aa on rRNA  Anticodons  (3 complementary nucleotides) GCA

Cracking the code  Francis Crick  Codons (Triplet code)-mRNA  Each codon corresponds to an aa  20 amino acids  Reading frame  Reading symbols in correct groupings

Cracking the code  1 or 2 deletions or additions  Gene was transcribed incorrectly  3 deletions  Reading frame would shift  Gene was transcribed correctly

WHYDIDTHEREDCATEATTHEFATRAT WHYIDTHEREDCATEATTHEFATRAT WHYDTHEREDCATEATTHEFATRAT WHYTHEREDCATEATTHEFATRAT

The code  Universal code  AGA codes for amino acid Arginine  Humans & bacteria  Genes from humans can be transcribed by mRNA from bacteria  Produce human proteins  Insulin

Protein synthesis DNA RNA Protein Transcription Translation

Prokaryotes  Transcription  Getting the code from DNA  Template strand  Strand of DNA that is transcribed or read  Transcribed RNA is complementary to the DNA

Prokaryotes  Coding strand  DNA strand not coded  Same sequence of nucleotides as the RNA transcript  Only T instead of U.

Prokaryotes  RNA polymerase  Enzyme  Adds nucleotides to the 3’end  5’to3’ direction  Does not need a primer to start

Prokaryotes  Stages of transcription  Initiation  Elongation  Termination

Prokaryotes  Initiation  Promoters:  Sequence on DNA where transcription starts  -35 sequence TTGACA  -10 sequence TATAAT  Sequences are not transcribed

Prokaryotes  RNA polymerase binds promoter  Unwinds DNA  Uses an ATP or GTP to start  Uses phosphate group  Transcription bubble:  RNA polymerase, DNA & growing RNA strand

Prokaryotes  Termination  Stop signal  Sequence on DNA  RNA transcript signals polymerase to detach from DNA  RNA strand separates from the DNA

Prokaryotes  Translation  Passing the code to make a polypeptide  mRNA binds to rRNA on the ribosome  mRNA attaches so only one codon is exposed at a time

Ribosome  Located in the cytoplasm  Site of translation  2 subunits composed of protein & RNA  Small (20 proteins and 1 RNA)  Large (30 proteins and 2 RNA)  3 sites on ribosome surface involved in protein synthesis  E, P, and A sites

Ribosome

Prokaryotes  tRNA (anti-codon)  Complementary sequence  Binds to mRNA  tRNA carries a specific amino acid  Adds to growing polypeptide  45 tRNA’s

Prokaryotes  Aminoacyl-t-RNA synthetases  Activating enzymes  Link correct tRNA code to correct aa  One for each 20 amino acids  Some read one code, some read several codes

Prokaryotes  Nonsense codes  UAA, UAG, UGA code to stop  AUG codes for start as well as methionine  Ribosome starts at the first AUG it comes across in the code

Prokaryotes  Translation  1. Initiation  2. Elongation  3. Termination

Prokaryotes  Initiation  Initiation complex  1. tRNA with formylmethionine attached binds to a small ribosome  2. Initiation factors position the tRNA on the P site  3. A site (aminoacyl) where other tRNA’s form

Prokaryotes  4. tRNA is positioned on to the mRNA at AUG  5. Attachment of large ribosomal unit

Prokaryotes  Elongation factors  Help second tRNA bind to the A-site  Two amino acids bind (peptide bond)  Translocation:  Ribosome moves 3 more nucleotides along mRNA in the 5’to 3’ direction

Prokaryotes  Initial tRNA moves to E site  Released  New tRNA moves into A site  Continues to add more aa to form the polypeptide

Prokaryotes  Release factors:  Proteins that release newly made polypeptides  Codon (UAG, UAA, UGA)  Release factor binds to the codon  Polypeptide chain is released from A site

Eukaryotes  Transcription (nucleus)  Initiation  Elongation  Termination

Eukaryotes  Initiation  Transcription Initiation Complex is formed  Transcription factors bind first to the promoter  RNA pol II binds DNA  Starts to transcribe

Fig. 17-7b Elongation RNA polymerase Nontemplate strand of DNA RNA nucleotides 3 end Direction of transcription (“downstream”) Template strand of DNA Newly made RNA 3 5 5

Fig. 17-UN1 Transcription unit Promoter RNA transcript RNA polymerase Template strand of DNA

Eukaryotes  Termination  Polyadenylation signal sequence  Recognized by RNA polymerase II  mRNA is released

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Eukaryotes  mRNA is modified  Nucleus  RNA processing

Eukaryotes  5’ cap  Addition of a GTP  5’ phosphate of the first base of mRNA  Methyl group is added to the GTP  3’poly-A-tail  Several A’s on the end of the mRNA

Eukaryotes  Introns:  non-coding sequences of nucleic acids  Exons:  coding sequences of nucleic acids

Euraryotes  RNA splicing  Cut out introns  Reconnect exons  snRNP’s (small nuclear RNA’s)  Spliceosome:  Many snRNP’s come together & remove introns

Eukaryotes  Translation  1. Initiating aa is methionine  2. Initiation complex is more detailed

Fig b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) mRNA binding site Large subunit Small subunit (b) Schematic model showing binding sites Next amino acid to be added to polypeptide chain Amino end Growing polypeptide mRNA tRNA EP A E Codons (c) Schematic model with mRNA and tRNA 5 3

Second nucleotide

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Fig. 17-UN3 mRNA Ribosome Polypeptide

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Similarities DNA RNA Protein Transcription Translation

Differences in gene expression  Transcription  1. Prokaryotes one RNA polymerase  Eukaryotes 3 RNA polymerases (poli-II mRNA synthesis)  2. Prokaryotes mRNA contain transcripts of several genes  Eukaryotes only one gene  3. Prokaryotes no nucleus so start translation before transcription is done

Differences in gene expression  3. Eukaryotes complete transcription before leaving the nucleus  4. Eukaryotes modify RNA Introns/exons  5. Prokaryotes Polymerase binds promoters  Eukaryotes transcription factors bind first then enzyme  6. Termination

Differences in gene expression  Translation  1. Prokaryotes start translation with AUG  Eukaryotes 5’cap initiates translation  2. Prokaryotes smaller ribosomes

Mutations  Changes in genetic information  Point mutations:  Change in a single base pair  Sickle cell mutation

Mutations  Two types  1. Base-pair substitution  Exchange one nucleotide and base pair with another  Silent mutations  No effect on proteins

Mutations  Missense mutations:  Substitutions that change one aa for another  Little effect

Mutations  Nonsense mutations  Point mutation codes for stop codon  Stops translation too soon  Shortens protein  Non-functional proteins

Mutations  2. Insertions or deletions  Additions or losses of nucleotides  Frameshift mutations  Improperly grouped codons  Nonfuctional proteins

Fig Wild-type 3 DNA template strand Stop Carboxyl end Amino end Protein mRNA A instead of G U instead of C Silent (no effect on amino acid sequence) Stop T instead of C A instead of G Stop Missense A instead of T U instead of A Stop Nonsense No frameshift, but one amino acid missing (3 base-pair deletion) Frameshift causing extensive missense (1 base-pair deletion) Frameshift causing immediate nonsense (1 base-pair insertion) Stop missing Stop Extra U Extra A (a) Base-pair substitution(b) Base-pair insertion or deletion

Mutagens  Chemical or physical agents  Mutations in DNA