Where are we? We covered. REPLICATION now.. TRANSCRIPTION + TRANSLATION

Transcription

1 Where are we? We covered REPLICATION now.. TRANSCRIPTION + TRANSLATION

2 Figure 14.4 Which is a Bacterial cell? Which is a Eukaryotic cell? What is transcription? What is translation? TRANSCRIPTION DNA Nuclear envelope RNA PROCESSING Pre-mRNA TRANSCRIPTION DNA mrna TRANSLATION mrna Ribosome TRANSLATION Ribosome Polypeptide Polypeptide We are going to focus mostly on Eukaryotic cell transcription and translation.

3 Figure 14.5 DNA template strand A C C A A A C C G A G T T G G T T T G G C T C A TRANSCRIPTION mrna U G G U U U G G C U C A TRANSLATION Codon Lets make some mrna! In Protein Eukaryotes we actually Trp we Phe first make Gly pre-mrna Ser or an RNA transcript or a primary RNA transcript or an immature RNA Amino transcript acid

4 Figure 14.9 DNA T A T A A A A A T A T T T T TATA box RNA polymerase II Promoter Transcription factors Start point Transcription initiation complex Nontemplate strand Template RNA transcript 1 2 A eukaryotic promoter Transcription starts at a start point within a big sequence called the promoter. Transcription unit=whole Transcription sequence being factors transcribed (starts at start point) 3 Several transcription factors bind to DNA. Transcription initiation complex forms.

5 Figure 14.9 DNA T A T A A A A A T A T T T T TATA box Promoter Transcription factors Start point Nontemplate strand Template strand 1 A eukaryotic promoter 2 Several transcription factors bind to DNA. RNA polymerase II Transcription factors 3 Transcription In Eukaryotes transcription factors bind initiation first and complex forms. trigger the making of the primary RNA transcript (or pre-mrna) Transcription initiation complex RNA transcript

6 Figure nucleotides are exposed to make a transcription bubble RNA polymerase Nontemplate strand of DNA RNA nucleotides RNA polymerase adds nucleotides to make new primary RNA transcript. It is single stranded! C C Newly made RNA A A T T U A C end C G C C G A A A A T T T U A Direction of transcription U Note: RNA has Uracil instead of Thymine!

7 Figure As the advancing wave of RNA synthesis takes place the new RNA molecule peels away from DNA template and helix reforms. 40 nucleotides per second! RNA polymerase C C Newly made RNA A A T T U A Nontemplate strand of DNA C end C G C C G A A A A T T T U A Direction of transcription RNA nucleotides U Template strand of DNA Uracil takes the place of Thymine

8 A single gene may have multiple transcription points and multiple RNA polymerases working on it like trucks in a convoy What does that mean for the product (the protein that is being made)?

9 Figure 14.4 Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA TRANSCRIPTION DNA mrna TRANSLATION mrna Ribosome TRANSLATION Ribosome Polypeptide Polypeptide

10 In Eukaryotes this initial RNA sequence is called pre-mrna or the primary transcript or the primary RNA transcript. It needs to go through post-transcriptional modification or processing before becoming a mature mrna! 1. Ends modified (5 cap, poly-a tail added) (Why? Seems to help export process, may reduce degradation at ends, may help ribosome grab.)

11 Figure Cap 1 30 Intron Intron Poly-A tail mrna Cap UTR Coding segment Poly-A tail UTR AAUAAA

12 2. Interior sections or introns cut out and exons kept and spliced together by spliceosome. This is called RNA splicing So.the mrna molecule that enters cytoplasm is a very abridged version!

13 Figure Cap 1 30 Intron mrna Intron Introns cut out and exons spliced together Poly-A tail Cap Coding segment Poly-A tail AAUAAA

14 RNA splicing is really amazing because.. A single gene can encode more than one kind of polypeptide or protein depends on which sections treated as exons! So protein products are much more diverse than number of genes.

15 Figure Cap 1 30 Intron mrna Intron Introns cut out and exons spliced together Poly-A tail Cap Coding segment Poly-A tail So lets say you were going to make a protein out of only two of the exons above how many AAUAAA different proteins could you potentially make?

16 Figure Why is this great for the organism? Exons DNA Troponin T gene Primary RNA transcript RNA splicing mrna or

17 Figure 14.4 Where does mrna go? Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA TRANSCRIPTION DNA mrna TRANSLATION mrna Ribosome TRANSLATION Ribosome Polypeptide Polypeptide

18 Figure 14.4b-3 While DNA stays safe and secure in nucleus, mrna takes the chances, venturing out into the cells cytoplasm and mingles with a whole new crew of construction enzymes and protein-making factories called ribosomes. TRANSCRIPTION RNA PROCESSING TRANSLATION (b) Eukaryotic cell mrna DNA Nuclear envelope Pre-mRNA Ribosome Polypeptide

19 Mature mrna is exciting but what we really want is a protein! To get a protein we need to make a polypeptide (a string of amino acids) THIS IS TRANSLATION (happens at ribosomes)

20 Figure 14.5 DNA template strand NOTE Uracil taking place of Thymine in mrna! A C C A A A C C G A G T T G G T T T G G C T C A TRANSCRIPTION mrna U G G U U U G G C U C A TRANSLATION Codon Protein Trp Phe Gly Ser Amino acid

21 Translation Figure 14.4b-3 mrna heads out into cytoplasm to attach to ribosome TRANSCRIPTION RNA PROCESSING DNA Nuclear envelope Pre-mRNA mrna TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

22 Figure Growing polypeptide trna molecules Exit tunnel E P A Large subunit Small subunit mrna P site (Peptidyl-tRNA What is a ribosome?? Exit tunnel binding site) E site (Exit site) mrna binding site (a) Computer model of functioning ribosome Small subunit (b) Schematic model showing binding sites Amino end Growing polypeptide Codons Next amino acid to be added to polypeptide chain Are tons of these in cytoplasm A site (AminoacyltRNA binding site) Actually made up of a kind of RNA (ribosomal E E P A trna RNA) Large mrna subunit Do you see mrna above? (c) Schematic model with mrna and trna

23 Figure 14.5 A focus DNA on codons! template Sets strand of three mrna A C nucleotides C A A A are C called C G A codons. G T At ribosome each codon will match with a particular T G G T T T G G C T C A a.a. TRANSCRIPTION mrna U G G U U U G G C U C A TRANSLATION Codon Protein Trp Phe Gly Ser Amino acid

24 Anther kind of RNA (transfer RNA or trna) is out in cytoplasm hanging around with amino acids! trna has an anticodon at one end and hooks onto a specific amino acid at other end if mrna codon is GGC trna anticodon to match will be CCG and would have grabbed CCG GGC

25 Figure Polypeptide Amino acids Trp Phe Ribosome trna with amino acid attached Gly trna mrna A A A U G G U U U G G C Codons Anticodon

26 More Translation. mrna-gets conveyed thru the ribosome unit until the start codon (Start codon is always AUG ) Start codon establishes reading frame (every set of three after that is a codon) Once hits start codon trna hauls appropriate amino acid to the ribosome

27 Figure 14.5 DNA template strand A C C A A A C C G A G T T G G T T T G G C T C A TRANSCRIPTION mrna U G G U U U G G C U C A TRANSLATION Codon Protein Trp Phe Gly Ser Amino acid

28 A little more detail about codons.. There are 64 possible codons. Why? [4 possible bases (A, G, C, U) and 3 bases per codon so 4 3 ] 3 are stop codons! (and one start codon..so actual is 64-4=60) BUT THERE are not 61 different amino acids Hmmmmm? In reality there are multiple codons that match each amino acid.

29 What is wobble? The idea that both AGA and AGG will both code for Arginine-3 rd slot is more flexible mrna Table GGU, GGC, GGA will also match to Glycine

30 Figure Growing polypeptide trna molecules Exit tunnel Too much detail! E P A Large subunit Small subunit mrna (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) E P A Exit tunnel A site (AminoacyltRNA binding site) Large subunit mrna Amino end E Growing polypeptide Next amino acid to be added to polypeptide chain trna mrna binding site Small subunit (b) Schematic model showing binding sites Codons (c) Schematic model with mrna and trna

31 Terminology! Polypeptide refers to a chain of amino acids Protein is typically the finished product-how do you get that finished product? FYI it is also called a mature protein.

32 Post Translational Modifications p285 Polypeptide starts to coil and fold due to its primary structure (its amino acid sequence) (might be a chaperone protein that helps it fold correctly) Groups are added (sugars, lipids, phosphate groups) Parts might be removed (e.g. amino acids from leading end or middle EX. Insulin is formed after a chunk of a.a. are taken out of its middle.) Polypeptides may be joined together to become subunits of a big protein like hemoglobin

33 Post Translational Modifications Once again we call the protein formed after these modifications a mature protein. Prokaryotes do this but not as much as Eukaryotes

34 Troubles with ends! (Not required material) During replication DNA polymerase can add only to 3 end so cannot complete 5 end. Repeated rounds of replication produce shorter and shorter DNA molecules with uneven ends. P259 txt RNA primer replacement is not a problem at origins of replication within the chromosome, because DNA polymerase I can attach to an "upstream" piece of DNA to backfill where the RNA primer was. At the ends there is no 3 piece of DNA available to use as a primer.

35 Figure (Not required Leading strand material) Lets assume this is the Overview Lagging Origin of replication strand Lagging strand Leading strand Overall directions end of the chromosome of replication 1 Primase makes RNA primer. Template 1. RNA comes off. strand RNA primer 2 DNA pol III 2. DNA pol I is for fragment 1 makes Okazaki fragment 1. supposed to add by hooking onto 3 piece of previous DNA 3 DNA pol III detaches. stretch-oops Okazaki fragment 1 RNA primer for fragment 2 Okazaki fragment 2 6 DNA ligase forms bonds between DNA fragments. 4 5 DNA pol III makes Okazaki fragment 2. DNA pol I replaces RNA with DNA. Overall direction of replication

36 Qs for Telomere Article and p 258 text! 1. What are telomeres? Do they contain genes? Do bacteria have telomeres? 2. What is telomerase and what does it do? What would happen in germ cells if telomerase did not exist? 3. Telomerase is not usually active in somatic cells, but turns on in germ cells, why? 4. Unusual activity of telomerase is often seen in what condition?

37 5. Non coding repetitive sequences? Apoptosis? What do these things mean? 6. What kinds of conditions are associated with shortened telomeres? 7. What did the researchers find? Can you make a sketch of the findings the way our textbook does for experiments it describes? 8. Look at Table 1. What are the numbers in the parentheses after the means! 9. Look at Figure 1. What does the little star on the line between the two elderly groups mean?

38 Error Bars! Cause and effect? Is telomere length a result of their being athletes? Or Are they athletes because they have long telomeres? (Perhaps long telomeres make their bodies work bezer, recover from workouts faster, means they are more likely to be athletes ) Figure 1. Telomere length expressed as T/S ra8o among athletes and non-athletes, stra8fied by age. Østhus IBØ, Sgura A, Berardinelli F, Alsnes IV, et al. (2012) Telomere Length and Long-Term Endurance Exercise: Does Exercise Training Affect Biological Age? A Pilot Study. PLoS ONE 7(12): e doi: /journal.pone hzp://

39 The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands. Rank the primers (the red specks) in the order they were produced. If two primers were produced at the same time, overlap them.

40 The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands. Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them. a and h then b and g then c and f and finally e and d

41 True of Leading strand, Lagging strand, or Both???? Daughter strand elongates away from replication fork Multiple primers needed Made in segments Made continuously Daughter strand elongates toward replication fork

42 True of Leading strand, Lagging strand, or Both???? Daughter strand elongates away from replication fork Lag Multiple primers needed Lag Made in segments Lag Made continuously Lead Daughter strand elongates toward replication fork Lead

43 In an analysis of the nucleotide composition of DNA, which of the following will be found? (Imagine counting number of nucleotides of each type) A = G and C = T G + C = T + A A = C A + C = G + T

44 In an analysis of the nucleotide composition of DNA, which of the following will be found? A = G and C = T G + C = T + A A = C A + C = G + T

45 Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine? 31% 42% 8% 16% It cannot be determined from the information provided.

46 Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine? 31% 42% 8% 16% It cannot be determined from the information provided.