1. Lecture 4: Transcription in Prokaryotes Chapter 6

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 transcription translation reverse transcription replication

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.

4. Transcription: How is an RNA strand synthesized? 1.Regulated by gene regulatory elements within each gene. 2.DNA unwinds next to a gene. 3.RNA is transcribed 5’ to 3’ from the template (3’ to 5’). 4.Similar to DNA synthesis, except: NTPs (nucleotide triphosphate) instead of dNTPs (no deoxy-) No primer No proofreading Adds Uracil (U) instead of thymine (T) RNA polymerase X

6. Prokaryotes possess only one type of RNA polymerase transcribes mRNAs, tRNAs, and rRNAs Transcription is more complicated in eukaryotes Eukaryotes possess three RNA polymerases: 1.RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S 2.RNA polymerase II, transcribes mRNAs and some snRNAs 3.RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs (RNA processing) *S values of rRNAs refer to molecular size, as determined by sucrose gradient centrifugation. RNAs with larger S values are larger/have a greater density.

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

8. RNA is a nucleic acid similar to DNA, but with critical functional differences 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. Most important: RNA can basepair with RNA or with single-stranded DNA, but it is normally single-stranded rather than double-stranded.

9. 07_05_RNA.jpg Single stranded RNA can fold into complicated 3D shapes resulting from intramolecular basepairing Structure of a ribozyme, an RNA enzyme Hairpin structures result from regions of sequence that are complementary to each other (inverted repeats).

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

11. Types of RNA

12. 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)

13. Three Steps to Transcription: 1.Initiation 2.Elongation 3.Termination Occur in both prokaryotes and eukaryotes. Elongation is conserved in prokaryotes and eukaryotes. Initiation and termination proceed differently.

14. 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.

15. Promoter and terminator sequences in bacteria

16. Step 1-Initiation, E. coli model ( Prokaryote ): Fig. 5.3 Each gene has three regions: 1.5’ Promoter, attracts RNA polymerase -10 bp 5’-TATAAT-3’ -35 bp 5’-TTGACA-3’ 2.Transcribed sequence (transcript) or RNA coding sequence 3.3’ Terminator, signals the stop point

17. How do we know where it starts? Answer: Consensus Nucleotide Sequence!

18. Step 1-Initiation, E. coli model: 1.RNA polymerase combines with sigma factor (a polypeptide) to create RNA polymerase holoenzyme Recognizes promoters and initiates transcription. Sigma factor required for efficient binding and transcription. Different sigma factors recognize different promoter sequences. 2.RNA polymerase holoenzyme binds promoters and untwists DNA Binds loosely to the -35 promoter (DNA is d.s.) Binds tightly to the -10 (transcription bubble) promoter and untwists *Note: RNA polymerase holoenzyme would fall off if promoter is not detected! ensures RNA polymerase to bind tightly to the DNA.

20. Step 2-Elongation, E. coli model: 1.After 8-9 bp of RNA synthesis occurs, sigma factor is released and recycled for other reactions. 2.RNA polymerase completes the transcription at 30-50 bp/second. 3.DNA untwists rapidly, and re-anneals behind the enzyme.

21. Step 3-Termination, E. coli model: - RNA polymerase halts and releases both the newly made RNA and the DNA template.

22. A termination signal consists of a string of A-T nucleotide pairs. RNA folds into a “hairpin” structure which helps to disengage the RNA transcript from the active site.

24. 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: 1/10 4 nucleotides DNA polymerase error rate: 1/10 7 nucleotides

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