Chapter 17 - From Gene to Protein

1. Chapter 17 - From Gene to Protein Now that we know how the genetic design information that codes for all the RNA/proteins necessary to build/maintain organisms is replicated so that it can be passed from cell to cell, organism to organism or even virus to virus…what is the next question?

2. Chapter 17 - From Gene to Protein The next question concerns how DNA… 1. …is replicated during S phase so that the information it encodes needed to build/maintain organisms can be passed to the next generation. 2. …stores this information that will be used to make all the RNA/polypeptides that will directly build/maintain the organism. molecular biology- the study of biology at the molecular level (overlaps biochemistry and genetics in particular). Much of what we have done thus far is molecular biology – cell resp, photosyn, membrane transport, endomembrane system, central dogma, etc… Mendelian genetics is not because you never discuss the molecular level, but chromosomal genetics is.

3. Chapter 17 - From Gene to Protein Your cells need “workers”. We have discussed many of these workers in detail at this point: glycolysis enzymes, krebs enzymes, ETC transporters, cytoskeleton, antibodies, insulin, carbonic anhydrase, hemoglobin, glucose transporter, Calvin enzymes, Photosystems, kinesin, Various receptors, signal transduction proteins, tRNA, ribosomes, photosystems, and the list goes on… What determines the structure/function of a protein/RNA? The sequence. What determines the sequence? The DNA (gene) sequence. What determines your DNA sequence? Your parents DNA sequence and the changes (mutations) that might have occurred between them and you…

4. Chapter 17 - From Gene to Protein NEW AIM: How is genetic information transmitted from DNA to protein? How is the genetic information transmitted from DNA to protein? ? Fig. 10.6A

5. Chapter 17 - From Gene to Protein NEW AIM: How is genetic information transmitted from DNA to protein? What did we call this process? ? Fig. 10.6A

6. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? What is the first step and what enzyme is involved? The Central Dogma of Molecular Biology ? Fig. 10.6A

7. Chapter 17 - From Gene to Protein NEW AIM: How is genetic information transmitted from DNA to protein? The Central Dogma of Molecular Biology Transcribe means to make a written copy. mRNA is a copy of a segment of DNA, a gene. They are the same language – nucleic acid language. By RNA polymerase …and the second step?

8. Chapter 17 - From Gene to Protein NEW AIM: How is genetic information transmitted from DNA to protein? The Central Dogma of Molecular Biology Translate means to convert between languages. In this case, nucleic acid language is translated into amino acid language by the ribosome and tRNA. By the ribosome and tRNAs

9. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? The Central Dogma of Molecular Biology Reminder (analogy): The nucleus is the library, the DNA/chromosomes are the reference books that cannot leave the library, and the mRNA is the transcription or copy of a small part of the DNA, a gene, that is slipped through the nuclear pore to a ribosome (rRNA + proteins) in the cytosol that will be involved in translating the nucleic acid language into amino acid language (a polypeptide) with the help of tRNA. Do bacteria have a library? They do not have a nucleus…transcription occurs in the semifluid (cytoplasm)

10. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Fig. 10.7 Reminder: A single chromosome has thousands of genes… Each gene codes for? A complementary piece of RNA (mRNA, tRNA or rRNA) If the gene codes for mRNA, then the mRNA will code for? A polypeptide Quaternary If the polypeptide is functional all by itself (no __________ structure), it is a…? Protein

11. Chapter 17 - From Gene to Protein NEW AIM: How is genetic information transmitted from DNA to protein? The Central Dogma of Molecular Biology

12. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) You be RNA polymerase and transcribe the above piece of DNA… Fig. 10.8B

13. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) PROBLEM: DNA has two strands. RNA polymerase only transcribes one strand into RNA… Which one? - That depends on the gene. The same strand will always be transcribed by RNA polymerase for a given gene. Fig. 10.8B

14. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) 5’ 3’ 5’ 3’ In this example, it is the top strand that will be transcribed. Transcribe it… Fig. 10.8B

15. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) 5’ 3’ 5’ 3’ SEEING DOUBLE: RNA polymerase will bind to the DNA, open up the strands (using ATP of course) and random RNA nucleotides (triphosphates) will bounce in and out of the active site until the complementary one bounces in and sticks long enough for the condensation reaction to occur forming a phosphodiester linkage. Which DNA strand does the transcribed strand look like? The RNA transcript will look like the non-transcribed strand with U substituted for T.

16. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) Template/antisense or non-coding strand The transcribed strand is also called the: 1. Template Strand Sense or coding strand 2. anti-sense strand 3. non-coding strand The reason for number one is obvious, but the other two are not...these are named this way because: The other DNA strand is called the: 1. Sense strand Why? Why? Because the sequence of this strand matches the RNA with U for T of course. Therefore, this DNA strand makes sense because it matches the RNA. Also, the RNA carries the CODE and therefore the strand it looks like is the CODE-ing strand. 2. Coding strand Fig. 10.8B

17. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? 1. TRANSCRIPTION (The Basics) 5’ 3’ Template/antisense or non-coding strand RNA polymerase is similar to DNA polymerase in that: 5’ Sense or coding strand 3’ It can only synthesize RNA from the 5’ to 3’ end… How would you label the DNA in this case? 5’ 3’ You label the sense strand the same way the RNA transcript is labeled and the complementary strand that RNA polymerase used to make the transcript must be antiparallel…

18. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 1: Write out the transcript of the following gene from 5’ to 3’ if the top strand is the sense strand. 5’ 3’ ATAGCGGCTATTA TATCGCCGATAAT 3’ 5’ ANS: 5’ AUUAUCGGCGAUA 3’ If the top strand is the sense strand then the template strand is the opposite strand or the bottom one. RNA polymerase can only make RNA 5’ to 3’ and therefore must start on the right and work toward the left looking at the bottom strand. You could also reason that the top is the sense and the transcript must read just like the sense from 5’ to 3’.

19. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 2: Write out the transcript of the following gene from 5’ to 3’ if the bottom strand is the antisense (non-coding) strand. 3’ 5’ ATAGCGGCTATTA TATCGCCGATAAT 5’ 3’ ANS: 5’ AUAGCGGCUAUUA3’ Since the bottom strand is the non-coding strand or antisense strand, this is the template. RNA polymerase looks at this one and adds the complementary bases starting at the 3’ end since it can only make RNA 5’ to 3’.

20. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 3: Write out the transcript of the following gene from 5’ to 3’ if the bottom strand is the sense (coding) strand. 3’ 5’ ATAGCGGCTATTA TATCGCCGATAAT 5’ 3’ ANS: 5’ UAAUAGCCGCUAU 3’ Since the bottom strand is the coding strand (sense strand), the top one is the template. RNA polymerase looks at the top strand and adds the complementary bases starting at the 3’ end since it can only make RNA 5’ to 3’.

21. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 4: RNA polymerase makes the following transcript: RNA Transcript: 5’ AUCGCGGUUACGG 3’ Draw out the piece of DNA corresponding to this transcript: 3’ 5’ ATCGCGGTTACGG TAGCGCCAATGCC 5’ 3’ You are given the transcript. There are a few ways to do this. I prefer thinking from the perspective of RNA polymerase. Since this is what it made, it must have looked at the complementary DNA strand going from 3’ to 5’, which I wrote as the bottom strand here. I then filled in the complementary DNA strand above it to complete the double stranded DNA molecule. DNA is always written with the 5’ end of one strand on the top left.

22. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 5: RNA polymerase makes the following transcript: RNA Transcript: 3’ AUCCGGCGAUUUCG 5’ Draw out the piece of DNA corresponding to this transcript: RNA Transcript (flipped over): 5’ GCUUUAGCGGCCUA 3’ 3’ 5’ GCTTTAGCGGCCTA CGAAATCGCCGGAT 5’ 3’ I will always write out the RNA transcript from 5’ to 3’ because this is how it is made and that is what makes sense to me. Then you finish it the same way as the previous one…

23. Chapter 17 - From Gene to Protein AIM: How is genetic information transmitted from DNA to protein? Question 6: You send in a segment of a gene to the DNA sequencing facility. They return the following sequence to you: 3’ 5’ GCAACTTCGCCATTAG This is the sense strand. What would the RNA transcript be? RNA Transcript: 5’ GCAACUUCGCCAUUAG 3’ It would be the same as the sense strand with U substituted for T.

24. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (the details) What parts of your genome (DNA/chromosomes) do RNA polymerases transcribe? The 30,000+ Genes How do the enzymes (RNA polymerases) “know” where the genes start and where they stop???

25. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (some details) a single gene We only need to look at how this works at a single gene as the process is similar for all of them.

26. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (some details) Let’s put this into some realistic context. Let’s imagine we are in the nucleus of a beta cell of your pancreas, which are the ones that secrete insulin when your blood glucose levels get too high (>140mg/dl). They need to be ready at any moment in case you drink a soda… and thus the gene is typically active and insulin is being made and packed into vesicles via the endomembrane system. The vesicles sit and wait for glucose to bind a receptor on the membrane followed by signal transduction, which will trigger the vesicles to fuse with the membrane and thus release the insulin into the blood. Let’s watch the mRNA being transcribed for the insulin gene… Fig. 10.9B

27. AIM: How is genetic information transmitted from DNA to Protein? (Transcription Unit) Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION Basic Anatomy of a Gene: 1. The Promoter – asequence of DNA that RNA polymerase will bind (“stick”) to indirectly with the help of other proteins called transcription factors in order to begin transcription (see video). a. In prokaryotes the consensus sequence is TATAAT and is called the Pribnow box b. In eukaryotes the consensus sequence is TATAAA and is called the TATA box 2. The Transcription Unit – the part that is transcribed into RNA (promoter and terminator are not transcribed) 3. The Terminator – sequence of DNA that will cause RNA polymerase to stop and fall off the DNA Fig. 10.9B

28. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION Let’s watch a video to see how these parts of the gene, RNA polymerase, a bunch of special protein called transcription factors and of course…ATP, come together to make transcription possible.

29. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION

30. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION of the gene has 3 general stages: A. Initiation i. RNA polymerase binds to promoter region ii. DNA unwinds and transcription begins (requires ATP) 5’ iii. The Promoter sequence “tells” RNA polymerase which strand of DNA to transcribe 3’ 5’ 3’ Fig. 10.9B

31. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION of the gene has 3 general stages: A. Initiation iv. Transcription factors - Additional proteins required for RNA polymerase to start transcription. - We have spoken many times about such factors being phosphorylated in the cytoplasm via signal transduction resulting in their export into the nucleus. ASIDE: ATP does NOT REDUCE anything, it phosphorylates. Fig. 10.9B

32. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION of the gene has 3 general stages: A. Initiation More Detail: Don’t memorize this level of detail unless you have nothing else to do. First email me though and I will find you something else to do. Fig. 10.9B

33. AIM: How is genetic information transmitted from DNA to Protein? Fig. 10.9B Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (3 stages) B. Elongation i. RNA polymerase polymerizes complementary RNA nucleotides across from the template/anti-sense/non-coding strand., which is always the same in a gene and is determined by the promoter. sense strand coding strand 5’ 3’ 5’ 5’ 3’ anti-sense strand non-coding strand 5’

34. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (3 stages) B. Elongation ii. Just like DNA polymerase, where does RNA polymerase get the energy to link together RNA nucleotides? A. From the nucleotides themselves: they are all triphosphates (ATP, GTP, UTP, CTP) and have a higher affinity for each other than for the diphosphate they are attached to… 5’ iii. Rate: ~60 nucleotides per second Fig. 10.9B

35. AIM: How is genetic information transmitted from DNA to Protein? Central Dogma (DNA to polypeptide) 1. TRANSCRIPTION (3 stages) C. Termination i. RNA polymerase reaches a sequence in the gene that causes it to fall off, releasing the completed RNA transcript. Fig. 10.9B

36. NEW AIM: How is genetic information transmitted from DNA to Protein? RNA polymerase making RNA (the red strand)

37. AIM: How is genetic information transmitted from DNA to Protein? What might be the evolutionary advantage of having a nucleus? After all, bacteria do not have nuclei and they make RNA and polypeptides from their chromosome similar to eukaryotes… Part of the answer might lie in RNA PROCESSING By separating the initial RNA transcript from the ribosomes in the cytoplasm, “workers” are able to modify the RNA in various ways…it is all about compartmentalization…

38. AIM: How is genetic information transmitted from DNA to Protein? RNA PROCESSING (eukaryotes ONLY) By separating the initial RNA transcript from the ribosomes in the cytoplasm, “workers” are able to modify the RNA in various ways…it is all about compartmentalization… 1. Adding the 5’ cap and the poly A (adenosine) tail

39. NEW AIM: How is genetic information transmitted from DNA to Protein? RNA Processing (eukaryotes) – the 5’ cap and poly A tail

40. NEW AIM: How is genetic information transmitted from DNA to Protein? 7-methyl-guanosine CAP

41. AIM: How is genetic information transmitted from DNA to Protein? RNA PROCESSING (eukaryotes ONLY) 1. Adding the 5’ cap and the poly A (adenosine) tail FUNCTION? A. Both appear to be required for nuclear export. B. Both protect the mRNA from hydrolysis in the cytoplasm by nucleases known as RNAses. C. The cap and tail assist the ribosome to bind 2. RNA Splicing

42. NEW AIM: How is genetic information transmitted from DNA to Protein? 2. RNA Splicing More detailed Anatomy of a Eukaryotic Gene: i. - Transcription unit of eukaryotes is broken into exons and introns. - The introns are named because they are “intervening” sequences. - Both the exons and introns are transcribed as shown, but… Fig. 10.10

43. NEW AIM: How is genetic information transmitted from DNA to Protein? 2. RNA Splicing ii. Introns are removed from the mRNA and the exons are SPLICED together by the spliceosome. -some = body iii. Spliceosomes are RNA and protein complexes…(what other complex is composed of RNA and protein, and is active between DNA and protein in the central dogma also supporting the RNA world hypothesis?) The ribosome Why do splicing??? Fig. 10.10

44. NEW AIM: How is genetic information transmitted from DNA to Protein? 2. RNA Splicing What is the spliceosome composed of? SnRNPs (“snurps”) 1. SnRNP = Small nuclear ribonucleoproteins (Small RNA/protein complexes in the nucleus) 2. Composed of a core snRNA molecule of ~150 nucleotides with associated proteins 3. Assorted SnRNPs combine to form the spliceosome Aside: Ribozymes Aside: Ribozymes are true RNA enzymes. Certain species have introns that splice themselves out (catalyze their own removal without help from a spliceosome). These are ribozymes.

45. NEW AIM: How is genetic information transmitted from DNA to Protein? Let’s look at a little history first… Beadle and Tatum (1941) In 1941, American geneticists Beadle and Tatum proposed the “one gene, one enzyme” hypothesis, which states that each gene codes for an enzyme (experiment is in your book…know it)…

46. NEW AIM: How is genetic information transmitted from DNA to Protein? Let’s look at a little history first… The hypothesis was later modified to the “one gene, one protein” hypothesis… It was again modified to the “one gene, one polypeptide” hypothesis… (you should know why) Getting closer and closer to the truth, but even this hypothesis is not always correct… because of ALTERNATIVE SPLICING

47. AIM: How is genetic information transmitted from DNA to Protein? ALTERNATIVE SPLICING Exons can be spliced together in different ways leading to different proteins/polypeptides being formed from the same gene… This may be one reason why splicing evolved – you can get more than one polypeptide per gene (not all genes do this).

48. AIM: How is genetic information transmitted from DNA to Protein? Exon Shuffling

49. NEW AIM: How is genetic information transmitted from DNA to Protein? RNA Splicing

50. NEW AIM: How is genetic information transmitted from DNA to Protein? The Final Mature mRNA: