Review: Proteins and their function in the early stages of replication

1. Review: Proteins and their function in the early stages of replication 4 3 2 1 1 = initiator proteins 2 = single strand binding proteins 3 = helicase 4 = topoisomerase (gyrase)

2. Replication • Two DNA polymerase enzymes are necessary for replication in E. coli • DNA polymerase I • DNA polymerase III

3. - Along each template DNA strand, leading and lagging strands can be observed. • - The names were suggested based on synthesis at any given region. • At any particular point in the DNA strand, if there is a leading • strand, the complementary strand will have lagging strand.

4. Replication • Two DNA polymerase enzymes are necessary for replication in E. coli • DNA polymerase I • DNA polymerase III • Both have polymerase and exonuclease activities (functions) • First let us take a look at the polymerase activity aspect of DNA polymerases and then discuss exonuclease activities

5. Replication • DNA Polymerase III • Synthesize new DNA in the 5’  3’ direction • Synthesizes long sequences of new DNA • Is highly processive; synthesizes DNA for a long period of time without releasing the template • For example, synthesizes leading strand • DNA Polymerase I • Synthesize new DNA in the 5’  3’ direction • Only synthesizes short sequences of new DNA • But before it could do this, it needs to remove RNA primers • This is achieved by its 5’  3’ exonuclease activity

6. 5’ 3’ exonuclease activity of DNA polymerase I

9. Replication • The phosphodiester backbone of adjacent DNA fragments must be joined after DNA synthesis by DNA polymerases I and III • This is done by the enzyme DNA ligase

10. Both DNA polymerases have proof reading activity This is a 3’  5’ exonuclease activity DNA Polymerase activity

12. Replication • DNA Polymerase I • Synthesize new DNA in the 5’  3’ direction • Only synthesizes short sequences of new DNA • 3’  5’ exonuclease activity (proofreading) • 5’  3’ exonuclease activity (remove primers) • DNA Polymerase III • Synthesize new DNA in the 5’  3’ direction • Synthesizes long sequences of new DNA • 3’  5’ exonuclease activity (proofreading) • NOTE: DNA polymerase III does not have the • 5’  3’ exonuclease activity

13. This week we will complete… Chapter 13 (transcription) Pages 348 – 361 Chapter 15 (translation) Pages 409 - 421

14. The Central Dogma(Francis Crick, 1958) (Transcription) (Translation) DNA  RNA  Protein (Gene/Genotype)(Phenotype) An informational process between the genetic material (genotype) and the protein (phenotype)

15. Properties of RNA RNA has the sugar ribose rather than deoxyribose

16. Properties of RNA Nucleotides carry the bases adenine, guanine and cytosine (like DNA) But uracil is found in place of thymine

17. Structure of RNA • Designate the Nucleotides • Purines • Guanine = G • Adenine = A • Pyrimidines • Uracil = U • Cytosine = C

18. Structure of RNA • Nucleotides join together, forming a polynucleotide chain, by phosphodiester bonds A phosphodiester bond A phosphodiester bond

19. Usually single-stranded

20. Can have a much greater variety of complex three dimensional shapes than double-stranded DNA

21. Classes of RNA for Transcription and Translation • Informational RNA (intermediate in the process of decoding genes into polypeptides) • Messenger RNA (mRNA) • Functional RNAs (never translated into proteins, serve other roles) • Transfer RNAs (tRNA) • Transport amino acids to mRNA and new protein • Ribosomal RNAs (rRNA) • Combine with an array of proteins to form ribosomes; platform for protein synthesis • Small nuclear RNAs (snRNA) • Take part in the splicing of primary transcripts in eukaryotes • Small cytoplasmic RNAs (scRNA) • Direct protein traffic in eukaryotic cells • Micro RNAs (miRNA) • Inhibits translation and induces degradation of complementary mRNA

22. RNA nucleotide sequences are complementary to DNA molecules New RNA is synthesized 5’ to 3’ and antiparallel to the template DNA template

23. DNA templateComplementary RNA Adenine Uracil Guanine Cytosine Cytosine Guanine Thymine Adenine Synthesized 5’ to 3’ and antiparallel to the template

24. Only one strand of the DNA acts as a template for transcription The template strand can be different for differentgenes But…. For each gene only one strand of DNA serve as a template

25. Transcription Catalyzed by the enzyme RNA polymerase

26. Single RNA polymerase (Prokaryotes) • Core enzymeHoloenzyme • 2 ,1 and 1 ’ subunits 2 , 1 , 1 ’ subunits plus • σ subunit • Polymerizes RNAFinds initiation sites

27. Initiation:The region that signals the initiation of transcription is a promoter

28. - 35 bases from initiation of transcription • Recognized by RNA polymerase • - 10 bases from initiation of transcription • Unwinding of DNA double helix begins here

29. Elongation: RNA is polymerized in 5’  3’ direction

30. Elongation • NTPS (ATP, GTP, CTP, UTP) are added • The energy is derived by splitting the high-energy triphosphate bond

31. Termination RNA polymerase recognizes signals (sequence) for chain termination Releases the RNA and enzyme from the template

32. Animation on Transcription • http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_1.html