Transcription & Translation

1. Transcription & Translation Biology 11 Chapter 17

2. The Central Dogma • The central dogma of biology reflects the flow of genetic information from DNA to protein • The central dogma of biology is not concerned with DNA replication, which is a completely separate event

3. Central Dogma of Biology • The general flow of genetic information is Protein mRNA DNA Translation Transcription Any DNA copied to any single stranded RNA molecule. There are three types of RNA molecules Process by which mRNA is Made into a functional protein

4. Regulation of Transcription • Translation is a costly event for a cell • Much GTP is used while making a protein • Transcription and translation are highly regulated events • A bacterial cell would not make an enzyme for breaking down lactose if not lactose was present • A liver cell will not make proteins or enzymes that only a nerve cell would need

5. Transcription in Bacteria • The conversion of DNA into an RNA transcript requires an enzyme known as RNA polymerase • RNA polymerase • Catalyzes the formation of a phosphodiester bond between the 3’ end of the growing mRNA chain and the new ribonucleotides • Transcription must occur in the 5’ to 3’ direction • No primer is needed to begin transcription

6. Transcription in Bacteria The template strand on DNA is what is “read” by RNA polymerase into a message. The non-template strand is confusingly referred to also as the coding strand because the DNA sequence in this strand is complementary to the message which is made into Protein.

7. RNA Polymerase Structure • X-ray crystallography indicates that • Large globular enzyme • Several channels in the interior • Core enzyme • Just RNA polymerase • Active site for catalysis • Holoenzyme • Sigma plus RNA polymerase Bacterial RNA Polymerase Holoenzyme

8. Initiation of Transcription in Bacteria • Promoters • Sequences of DNA which “inform” RNA polymerase where to begin transcription • Located on non-template strand • 40 to 50 base pairs in length • All have a TATAAT sequence • -10 box centered 10 bases away from the site where transcription begins (+1) • All have a TTGACA • -35 box located 35 bases away from the +1 site • Sequences inside promoter but outside -10 or -35 vary widely among genes

9. Terms • Downstream • DNA located in the direction in which RNA polymerase will transcribe • Upstream • DNA located in the opposite direction in which RNA polymerase will transcribe Upstream Downstream Promoter TTGAC TATAAT Gene -35 -10 +1

10. Initiation of Transcription in Bacteria • Sigma makes the initial contact with DNA that starts transcription in bacteria • Once sigma has bound to the DNA the helix opens and the template is threaded through a channel • NTPs enter a different channel and diffuse to the active site • Incoming NTPs base pair complementary with the template strand • A base pairs with U not T

13. Opens the helix Steers the template and non-template strands into the correct channels

14. Termination of Transcription in Bacteria • DNA sequence functions as a transcription termination signal • Makes a stretch of RNA which as soon as it is synthesized can base pair with itself • Hairpin loop – RNA secondary structure • The formation of the hairpin disrupts the interaction between RNA polymerase and the RNA transcript resulting in the physical separation

15. Termination of Transcription in Bacteria

16. Transcription in Eukaryotic Cells • Transcription of eukaryotic cells is more complex and requires more proteins and regulators • Transcription factors • The initiation of transcription is accomplished by basal transcription factors • Function analogous to sigma • Can interact with DNA independently of RNA polymerase

18. Transcription in Eukaryotic Cells • Eukaryotic cells have three distinct types of RNA polymerases discovered by accident while working with the death cap mushroom • RNA pol I • Transcribes large rRNA subunit of ribosomes • RNA pol II • Transcribes mRNA, snRNPs • RNA pol III • Transcribes small rRNA subunit, tRNA and snRNPs

19. Death Cap Mushroom • While working out the mechanism of action for the α-amanitin toxin biologist discovered that cells exposed to the toxin contained no mRNA • The production of rRNA and tRNA continued • At higher concentrations tRNA production stopped as well • Based on this data what does α-amanitin toxin block?

20. Transcription in Eukaryotic Cells • Eukaryotic genes contain promoters • Promoters are much more diverse and complex • Many promoters recognized by RNA pol II include a TATA box at -30 • Some promoters recognized by RNA pol II do not contain a TATA box • RNA pol I and RNA pol III interact with entirely different promoters

21. Transcription in Eukaryotic Cells • Unlike prokaryotic cells eukaryotic cells must process the mRNA in the nucleus before it can be made into a protein • Three major modifications are made • Intron splicing • Addition of poly A tail • Addition of 5’ cap

22. The Discovery of Introns • In the 1970’s Phillip Sharp and colleagues took a fragment of DNA and hybridized it to a corresponding RNA fragment • They expected to find under the magnification of an electron microscope a DNA/RNA hybrid molecule which matched up exactly • Instead they found single stranded looped regions which extended out from the hybird

24. Exons & Introns • Exons • Coding regions of DNA • Expressed as proteins • Introns • Non-coding regions of DNA • Intervening

25. Introns Are No Small Matter

26. Intron Removal • Introns are removed in a process known as intron splicing • Splicing occurs while transcription is still underway • Removed by ribozymes known as small nuclear ribonucleoproteins • snRNPs • The boundaries of introns are precisely defined

27. Intron Removal E1 Intron 1 Intron 2 E2 E3 E3 E1 E2

28. GU AG

30. Intron Splicing: Reaction mechanism in detail

31. Adding a Cap and a Tail • 5’ cap • 7 methylguanylate with three phosphate groups • Recognition signal for translation machinery • Protection from exonucleases • Poly A tail • 250 A’s added by enzyme in the nucleus • Protection from degradation by ribonucleases in the cytoplasm

32. Comparing Transcription: Bacteria vs. Eukaryotes

33. Ribosomes: Site of Protein Synthesis In both prokaryotes and eukaryotes protein synthesis takes place on the ribosomes in the cytoplasm. Prokaryotic cells: Transcription and translation are simultaneous Eukaryotic cells: Transcription and translation are spatially and temporally separated

34. Mechanism of Protein Synthesis • Recall from chapter 15 that a codon codes for the correct amino acid • One early hypothesis was that the amino acids and the codons interact directly • Francis Crick pointed out that the chemistry involved did not make sense • How could a hydrophobic amino acid side group hydrogen bond and interact with a nucleic acid

35. Mechanism of Protein Synthesis • Crick suggested that some sort of adapter molecule holds amino acids in place while interacting with the mRNA codon

36. tRNA are the Adaptor Molecules • tRNA – transfer RNA • Molecule that becomes covalently linked to an amino acid • Also known as an aminoacyl tRNA • To become charged (carry an amino acid) requires an input of ATP and enzymes known as aminoacyl tRNA synthetases

37. Aminoacyl tRNA Synthetases • Responsible for catalyzing the addition of amino acids to tRNA • For each of the 20 amino acids there is a different aminoacyl tRNA which can bind to one or more tRNA

40. What Happens to Amino Acids on tRNA’s

41. What Happens to Amino Acids on tRNA’s In order to make this graph researches had to isolate the cytoplasmic populations of tRNA and protein at two time points and then analyze each fraction for concentration of radioactivity tRNA Protein

42. Structure of tRNA Anticodon In vivo 3D structure Secondary structure with hairpin loops

43. How Many tRNAs Are There? • Recall from chapter 15 that 61 codons code for 20 amino acids • Three codons code for stop • Are there 61 tRNA in a cell • NO! • Most cells contain about 40 tRNAs • How can 61 codons be translated with only 2/3 of the tRNAs?

44. Wobble Hypothesis • Francis Crick proposed the wobble hypothesis • Non-standard base pairing at the third position allows for a limited flexibility in base pairing • Many amino acids are specified by more than one codon • Codons of the same amino acid have the same nucleotides at the first and second positions • CAA and CAG code for glutamine • The anticodon GUU can base pair with both CAA and CAG

45. Met Anticodon Three nucleotide unit of tRNA Binds to codon on mRNA Small rRNA subunit UAC Codon Three nucleotide unit of mRNA How a Protein is Made AUG

46. Thr Small rRNA subunit UAC UGA New peptide bond made After the first peptide Bond has been made (catalyzed by the rRNA) the entire unit will Shift down the message to the next codon Met CCU CCC UUU UAG ACU AUG

47. Small rRNA subunit UGA Met Thr Pro Phe Gly GGA UAC GGG CCC UAG CCU ACU AUG UUU

48. All the Players of Translation

49. Translation • Initiation • Bacteria • mRNA region known as the ribosome binding site or Shine-Dalgarno sequence binds to small rRNA subunit • Eukaryotes • Initiation factors known bind to 5’ cap and guide it to the ribosome • Elongation • Peptide bond formation using ribozyme • Termination • Releasing factor binds to STOP codon • Catalyzes the hydrolysis of bond between tRNA in P site and peptide change