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Protein Synthesis

Protein Synthesis. A c loser look at how your body makes proteins!. Journal. What sequence of amino acids would the following DNA strand make? DNA strand = T A C A C C C G A A G T G T C G C A G A G A C T. Transcription. RNA Polymerase

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Protein Synthesis

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  1. Protein Synthesis A closer look at how your body makes proteins!

  2. Journal • What sequence of amino acids would the following DNA strand make? • DNA strand = T A C A C C C G A A G T G T C G C A G A G A C T

  3. Transcription • RNA Polymerase • Opens DNA strand & begins to transcribe the transcription unit into RNA (no primer needed) • Copies 5’ – 3’ (same as DNA replication) • RNA Pol. attaches to promoter sequence of gene & ends at terminator (in bacteria) • Terminator is downstream of promoter

  4. Synthesis of rna transcript • 3 stages: • Initiation • Elongation • Termination Initiation - promoter includes start point = where RNA synthesis begins • transcription factors (proteins) mediate binding of RNA pol. (in eukaryotes) = transcription initiation complex  includes TATA box

  5. Synthesis of rna transcript 2. Elongation - RNA pol. travels along strand - RNA strand peels away from DNA - DNA reforms **OCCURS AT SPEED OF 40 NUCLEOTIDES PER SECOND!! Can make protein in large amounts = many RNA poly. follow each other down DNA strand • 3 stages: • Initiation • Elongation • Termination

  6. Synthesis of rna transcript • 3 stages: • Initiation • Elongation • Termination 3. Termination EUKARYOTES RNA pol codes for polyadenylation signal (AAUAAA) = causes pre-mRNA strand to detach BACTERIA Terminator on DNA strand with termination sequence that triggers RNA pol. to detach

  7. RNA PROCESSING (euk. Cells) • Enzymes modify RNA before sent to cytoplasm •  during RNA processing, RNA goes through ALTERATIONS and segment SPLICING ALTERATION of mRNA ends: 5’ end receives a 5’ cap = modified G 3’ end receives a poly-A tail (50-250 more A nucleotides) IMPORTANCE? Facilitate in export of mRNA Protect mRNA from degradation by hydrolytic enzymes Help ribosome attach to 5’ end to begin translation

  8. RNA PROCESSING (euk. Cells) • Enzymes modify RNA before sent to cytoplasm •  during RNA processing, RNA goes through ALTERATIONS and segment SPLICING RNA SPLICING: *average length of transcription unit is 27,000 nuc. HOWEVER… it only takes 1,200 nuc. in RNA to code for the average-sized protein of 400 a.a. These non-coding segments are intermixed with coding segments … SO coding segment of DNA is not continuous! Noncoding segments are called INTRONS while the coding segments are called EXONS b/c they are expressed How are these introns recognized and spliced out?

  9. Enter the spliceosome! • Small nuclear ribonucleoproteins (snRNPs – pronounces “snurps”) •  made of snRNA and proteins •  located in cell nucleus and recognize splice sites in RNA •  several snRNP’s join together to form a spliceosome (almost as big as a ribosomes!) • Spliceosomes splice introns and bind remaining exons together

  10. Alternative rna splicing • Many genes can give rise to more than one protein  depends on what segments are treated as exons vs. introns • EX: sex differences in fruit flies it largely due to differences in how males and females splice genes • What would occur if a few introns were left in the sequence?

  11. Only 20 amino acids in eukaryotic cell! Translation • OVERVIEW: mRNA read in the ribosome (rRNA) by tRNA which delivers corresponding amino acids to the growing polypeptide chain • Let’s take a look • at the players!

  12. Transfer RNA (tRNA) • Variety • Only carry 1 amino acid and 1 anticodon • tRNA binds with correct amino acid with help of enzymes called aminoacyl-tRNAsynthetases  these synthetases are specific to an amino acid so there are a total of 20 different synthetasesin cytoplasm • tRNA recognition of codons is not as strict for final base = wobble EX: tRNA anticodon UCU can base pair with codons AGA and AGG, both code for arginine

  13. Take some time to answer the following questions: • What are amino acids? • What are a chain of Amino Acids called? • How many different Amino Acids are there? • How do you determine what amino acids the gene is coding for? • Codon vs. Anticodon? • Start codon vs. stop codon?

  14. Protein Synthesis Review

  15. Review protein Synthesis

  16. Only 20 amino acids in eukaryotic cell! Translation • OVERVIEW: mRNA read in the ribosome (rRNA) by tRNA which delivers corresponding amino acids to the growing polypeptide chain • Let’s take a look • at the players!

  17. Transfer RNA (tRNA) • Variety • Only carry 1 amino acid and 1 anticodon • tRNA binds with correct amino acid with help of enzymes called aminoacyl-tRNAsynthetases  these synthetases are specific to an amino acid so there are a total of 20 different synthetasesin cytoplasm • tRNA recognition of codons is not as strict for final base = wobble EX: tRNA anticodon UCU can base pair with codons AGA and AGG, both code for arginine

  18. Ribosomes • Consists of 2 subunits (1 large and 1 small) made of proteins and rRNA • Subunits made in nucleolus that come together when mRNA is read • Each ribosome has 3 sites for tRNAs to attach • P site (peptidyl-tRNA) = holds tRNA carrying the growing poly peptide chain • A site (aminoacyl-tRNA) =holds the tRNA carrying the next amino acid to be added to the chain • E site (exit) = discharges “empty” tRNA molecules • MUCH evidence supports that rRNA catalyzes the translation reaction NOT the protein that make up the ribosome • DID YOU KNOW… rRNA is the most abundant RNA in cells?

  19. 3 stages: Initiation Elongation Termination Building a polypeptide • Initiation • - small ribosomal subunit binds to mRNA and tRNA begins scanning mRNA at 5’ cap until it hits AUG (start codon) when translation begins  establishing the reading frame for translation • - large ribosomal subunit binds, completing initiation complex 2. Elongation • -amino acids are added one by one • - the mRNA is moved through ribosome in one direction only: 5’ first •  importance: ribosome and mRNA move relative to one another codon by codon 3. Termination • - occurs when a STOP codon (UAG, UAA, UGA) is reached • - a release factor is binds to stop codon and adds a water molecule instead of an amino acid to polypeptide chain and releases chain fromtRNA/ribosomal complex The elongation cycle takes less than a tenth of a second in bacteria!

  20. Polyribosomes DID YOU KNOW…? • A single mRNA can be copied multiple times AND copied by multiple ribosomes at the same time! • String of ribosomes copying the same mRNA strand = polyribosomes

  21. Mutations Let’s try identifying the different types of mutations! • Good or bad? • Types of Mutations: • Point mutation – changes to a single nucleotide (pic sickle cell) • A. Substitution – replacement of one nucleotide and its partner with another pair CAN BE A:  silent mutation = no change to amino acid sequence  missense mutation = change to amino acid sequence  nonsense mutation = change in amino acid yields an early stop *leads to shorter polypeptide *most nonsense mutations lead to nonfunctional proteins 2. Insertions and deletions – add or lose nucleotide pairs in a gene - have the WORST affect on proteins b/c they shift the reading frame which cause this type of mutation to be a FRAMESHIFT MUTATION

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