Central dogma II -- Transcription

1. Central dogma II -- Transcription • DNA -> RNA -> protein • But for retroviruses, DNA can also be made by reverse transcription of RNA. • To understand the lifecycle of a retrovirus, we need to know more about how DNA is replicated and transcribed, and how RNA is translated into protein.

2. Vocabulary • Replication -- copying DNA before cell division • Transcription -- making an RNA copy (messenger RNA or mRNA) of DNA. Note -- Transcription involves copying in the same language (e.g., court transcription). • Translation -- making a protein from the mRNA. Note -- The nucleic acid language is being translated into the protein language. DNA ---------> DNA DNA ---------> RNA ----------> Protein RNA ---------> DNA replication transcription translation reverse transcription

3. Transcription: DNA --> RNA • DNA replication ensures that genetic information is passed on unchanged from a cell to its descendents. • The major thing cells do with genetic information is use it to encode PROTEINS. • Every cell contains all of an organism’s genes, so each cell could (in theory) make every protein. But which proteins are made is determined by which genes are copied into RNA.

4. Cells express appropriate levels of proteins by regulating transcription and translation Two amplification steps: one gene can make many RNA copies, and one RNA can be translated into many proteins.

5. RNA is a nucleic acid similar to DNA, but with critical functional differences Most important: RNA can basepair with RNA or with single-stranded DNA, but it is normally single-stranded rather than double-stranded. The sugar in RNA contains an additional OH group, so is ribose, not deoxyribose. RNA uses uracil instead of thymine. Uracil can still basepair with Adenine.

6. Single stranded RNA can fold into complicated 3D shapes resulting from intramolecular basepairing 07_05_RNA.jpg Structure of a ribozyme, an RNA enzyme Hairpin structures result from regions of sequence that are complementary to each other (inverted repeats).

7. Clicker question When complementary RNA bases in an inverted repeat pair with each other, they form which type of structure? 1) Parallel double helix 2) Anti-parallel double helix 3) Parallel b-sheet Anti-parallel b-sheet -helix 6) Any of the above are possible

8. Clicker question When complementary RNA bases in an inverted repeat pair with each other, they form which type of structure? 1) Parallel double helix 2) Anti-parallel double helix 3) Parallel b-sheet Anti-parallel b-sheet -helix 6) Any of the above are possible

9. Clicker question 3D structures of RNA are easier to predict than 3D structures of proteins. 1) True 2) False

10. Can design and make DNA-based origami using single stranded DNA: http://www.dna.caltech.edu/~pwkr/ Giant DNA complexes, imaged with an atomic force microscope (measures surface topography). False color used to indicate height. Scale: 100s of nanometers.Images taken from Paul Rothemund’s website: http://www.dna.caltech.edu/~pwkr/ • Many web-available structure prediction programs • e,g., http://www.santafe.edu/~pth/rna.html Base-pairing makes prediction of RNA structure reasonably accurate

11. The predictability of Watson-Crick base pairing allows design of DNA-based digital logic circuits Erik Winfree’s lab at Caltech has designed and demonstrated function of AND, OR, and NOT gates, signal restoration, amplification, feedback and cascading. We may use DNA circuits someday instead of, or in addition to, silicon-based electronics. Seelig et al., 2006, “Enzyme-free Nucleic Acid Logic Circuits, Science 314: 1585-1588. See webpage for the Molecular Programming Project www.ist.caltech.edu/mpp/

12. Note that RNA polymerase unwinds a short region of the DNA double helix. DNA is transcribed into RNA by the enzyme RNA polymerase

13. Clicker question This electron micrograph shows many molecules of RNA polymerase simultaneously transcribing two adjacent genes. The RNA polymerase molecules are moving Right to left Left to right Not enough information to determine

14. “Nearly every major process in a cell is carried out by assemblies of 10 or more protein molecules… each of these protein assemblies interacts with several other large complexes… Indeed, the entire cell can be viewed as a factory that contains an elaborate network of interlocking assembly lines, each of which is composed of a set of large protein machines.”Bruce Alberts, Cell 92:291 (1998)

16. Eukaryotic gene transcription -- Activation begins at a distance

17. Cramer, P. et al. (2000) Science 288:640

18. Transcription video from DNA interactive

19. Clicker question It normally takes a large amount of free energy to separate DNA strands -- e.g., you would need to heat DNA to nearly boiling temperature in the lab to separate strands. RNA polymerase works at 37˚C, so how is it able to unwind the DNA in a closed circular bacterial chromosome while it transcribes? 1) RNA polymerase has an associated helicase, which uses the energy of ATP to force the DNA to unwind. 2) The RNA-DNA helix has a lower free energy than a DNA helix, so the formation of the RNA-DNA helix displaces the other DNA strand. 3) RNA polymerase degrades one of the DNA strands, so that the remaining strand can pair with the RNA strand. 4) Bacterial DNA is negatively supercoiled (i.e., underwound so that it has more than 10.4 basepairs/turn), therefore unwinding a short stretch of double helical DNA is energetically favorable.

20. To convert between 1 & 2 requires breaking and rejoining of a bond. This can be accomplished by a topoisomerase enzyme. No breaking of bonds is required to convert between 2 & 3. Double stranded DNA can wrap around itself to form supercoiled structures 2 1 3 Change in the linking number No change in the linking number The topology of DNA is studied by mathematicians. http://en.wikipedia.org/wiki/DNA_supercoil

21. RNA polymerase II movie Roger Kornberg received the 2006 Nobel Prize in Chemistry for studies on the “molecular basis of eukaryotic transcription”.

22. Comparison of RNA and DNA polymerases • Both catalyze similar chemical reactions: formation of phosphodiester bonds linking nucleotides. • RNA polymerase links ribonucleotides. Final product is single-stranded RNA. • DNA polymerase links deoxyribonucleotides.Final product is double-stranded DNA. • RNA polymerase error rate: 1/104 nucleotidesDNA polymerase error rate: 1/107 nucleotides

23. Which is potentially most serious, errors made by DNA polymerase RNA polymerase Ribosome Bi1 students Bi1 TAs Bi1 professors

24. Three major types of RNA • mRNA (messenger RNA) codes for proteins. • rRNA (ribosomal RNA) forms part of the ribosome, a machine involved in translation of RNA into protein. (Next lecture) • tRNA (transfer RNA) binds amino acids and bring them to the ribosome during protein translation. (Next lecture)

25. Signals in DNA tell RNA polymerase where to start and stop transcription • Synthesis starts at a promoter, a conserved sequence 5’ of a gene. • Chain elongation occurs until RNA polymerase encounters a termination sequence on the DNA. • RNA polymerase releases the single stranded RNA and the double stranded DNA template upon encountering a termination sequence. • This slide depicts a bacterial RNA polymerase. Transcription initiation in eukaryotic cells is more complicated.

26. Promoter and terminator sequences in bacteria

27. Clicker question Propose a mechanism by which the highlighted areas could cause transcription to stop. 1) The highlighted sequences contain stop codons. 2) RNA polymerase stops at pink sequences. 3) The highlighted sequences contain DNA mismatches, which destabilize RNA polymerase. 4) The first highlighted sequence contains a binding site for a termination factor, which knocks RNA polymerase off the DNA. 5) The first two sequences are inverted repeats of each other, thus a hairpin, which is a signal for termination.

28. Clicker question Propose a mechanism by which the highlighted areas could cause transcription to stop. 5) The first two sequences are inverted repeats of each other, thus a hairpin, which is a signal for termination.