Chapter 8 Lecture Outline. Transcription, Translation, and Bioinformatics

Transcription

1 Chapter 8 Lecture Outline Transcription, Translation, and Bioinformatics

2 Replication, Transcription, Translation n Repetitive processes Build polymers of nucleotides or amino acids n All have 3 major steps Initiation n Bind polymerizing machine, first monomer to template DNA polymerase, RNA polymerase, ribosome Elongation n Read template, add next monomer DNA, RNA, Protein Termination n Release machine and completed product 2009 W. W. Norton & Company, Inc. 2

3 RNA Polymerase, Sigma Factor n RNA Polymerase is a large molecular machine 4 proteins in one complex n α, β, βʹ, ω Binds DNA, reads sequence n Polymerizes RNA n Where on DNA to bind? Sigma (σ) factor n Smaller protein n Guides RNA polymerase to target DNA sequence Promoter 2009 W. W. Norton & Company, Inc. 3

4 Transcription Initiation n Sigma factor binds core RNA polymerase Forms RNA polymerase holoenzyme n RNA polymerase binds promoter Sequence on DNA 10 and 35 bases upstream of start site n Polymerase unwinds DNA at promoter Open complex n Sigma factor released 2009 W. W. Norton & Company, Inc. 4

5 Transcription Elongation n Core polymerase adds RNA to 3ʹ end Energy for base addition comes from base n mrna(n) + NTP mrna(n+1) + diphosphate Added base is complementary to template strand n Non-template strand also complementary to template n mrna has same sequence as non-template strand 5 U instead of T mrna non-template strand direction of movement template strand core polymerase 2009 W. W. Norton & Company, Inc. 5

6 Transcription Termination n Polymerase slows at pause site GC-rich sequence, forms stem loop n Rho-dependent termination Rho (ρ) factor binds to mrna n Slides along mrna to polymerase n Breaks polymerase, mrna off of DNA 2009 W. W. Norton & Company, Inc. 6

7 Transcription Termination n Rho-independent termination Series of U residues downstream of pause site n DNA-RNA UA base pairs are least stable Even less stable if polymerase is stalled n mrna breaks off of DNA, polymerase released 2009 W. W. Norton & Company, Inc. 7

8 Types of RNA n mrna messenger n rrna ribosomal n trna transfer All used to carry information from DNA to protein n Other RNAs regulate transcription snrna small nuclear: n mrna splicing in eukaryotes, archaea mirna micrornas n Regulate transcription in eukaryotes n Other types 2009 W. W. Norton & Company, Inc. 8

9 trna n Set of trnas bind individual amino acids trnas have specific shape trnas have 3-base anticodon n Base pair to codons in mrna 61 codons 20 amino acids 3 codons signal Stop n Proteins add amino acid to trna Aminoacyl-tRNA transferase Requires energy of ATP AMP 2009 W. W. Norton & Company, Inc. 9

10 The Genetic Code 2009 W. W. Norton & Company, Inc. 10

11 Ribosome = Protein Polymerase n Very large molecular machine 2 subunits, 52 proteins, 3 rrnas n Mostly rrna Can bind 1 mrna + 3 trnas Ribosomal large (50s) subunit Proteins rrna 2009 W. W. Norton & Company, Inc. 11

12 Translation of RNA to Protein n Initiation Initiation factors bind ribosome to 1st codon, AUG n Elongation EF-Ts, EF-Tu, EF-G bring GTP energy n Polymerization, movement of ribosome along mrna n Termination Releasing factors undock ribosome from mrna 2009 W. W. Norton & Company, Inc. 12

13 Coupling of Transcription and Translation n Transcription creates mrna Multiple mrnas made from a single gene n Ribosomes bind mrna While mrna is still being created Multiple proteins made rapidly from each mrna n Advantage of not having a nucleus 2009 W. W. Norton & Company, Inc. 13

14 Protein Modification n Enzymes modify translated protein fmet removed from N-terminus Small groups added to amino acids n Phosphoryl groups added n Methyl groups added n Adenylate groups added Protein may be cleaved Protein may be refolded by helping enzymes 2009 W. W. Norton & Company, Inc. 14

15 Protein Folding n Protein structure determined by Amino acid sequence n Many proteins fold spontaneously Chaperones n GroEL-GroES Complex Refolds denatured proteins Uses ATP Barrel shape Protein to be refolded fits into center n DnaK protein 2009 W. W. Norton & Company, Inc. 15

16 Protein Traffic n Many bacterial proteins reside in cytoplasm n Others targeted to other sites Plasma membrane Periplasm Gram - outer membrane Secreted outside of bacterium n Signal sequence Targets protein for transport 2009 W. W. Norton & Company, Inc. 16

17 Protein Secretion n Signal sequence N-terminal amino acids Bound by signal recognition particle (SRP) Targets ribosome to plasma membrane n Some proteins enter plasma membrane directly 2009 W. W. Norton & Company, Inc. 17

18 Protein Secretion n Sec-dependent pathway Delivers proteins to periplasm n Type I secretion system Secretes protein out of bacterium n 5 other secretion systems known 2009 W. W. Norton & Company, Inc. 18

19 Protein Degradation n All organisms constantly rebuild themselves Cells die, are replaced n All cells constantly rebuild themselves Cytoskeleton is rebuilt Cell wall grows and is remodeled Proteins are degraded when not needed n Amino acids reused 2009 W. W. Norton & Company, Inc. 19

20 Protein Degradation n Proteins survive for minutes to days Sequence, shape, function determine half-life n Proteases cut proteins Cut at specific amino acid sequences n Proteasomes degrade proteins Barrel shape n Eukaryotes add signal to proteins Ubiquitin tag causes degradation 2009 W. W. Norton & Company, Inc. 20