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Protein Synthesis

Protein Synthesis. Genome - the genetic information of an organism DNA – in most organisms carries the genes RNA – in some things, for example retroviruses like the AIDS virus Gene - a DNA sequence that is transcribed (includes genes that do not encode proteins).

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Protein Synthesis

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  1. Protein Synthesis • Genome - the genetic information of an organism • DNA – in most organisms carries the genes • RNA – in some things, for example retroviruses like the AIDS virus • Gene- a DNA sequence that is transcribed (includes genes that do not encode proteins)

  2. Information specifying protein structure • Informationflow: CENTRAL DOGMA • DNARNAPROTEIN • Transcription - copying of the DNA sequence information into RNA • Translation - Information in RNA molecules is translated during polypeptide chain synthesis

  3. Biological information flow

  4. Types of RNA • (1) Transfer RNA (tRNA) • Carries amino acids to translation machinery • Very stable molecules • (2) Ribosomal RNA (rRNA) • Makes up much of the ribosome • Very stable, majority of cellular RNA • (3) Messenger RNA (mRNA) • Encodes message from DNA to ribosomes • Rapidly degraded by nucleases

  5. Biological information flow

  6. RNA Polymerase • RNA polymerase (RNA pol) catalyzes DNA-directed RNA synthesis (transcription) • RNA pol is core of a larger transcription complex • Complex assembles at one end of a gene when transcription is initiated • DNA is continuously unwound as RNA pol catalyzes a processiveelongation of RNA chain

  7. The Chain Elongation Reaction • Mechanism almost identical to that for DNA polymerase • Growing RNA chain is base-paired to DNA template strand • Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template • New phosphodiester bond formed, PPi released

  8. Catalyzes polymerization in 5’ 3’ direction • Is highly processive, and thermodynamically assisted by PPi hydrolysis • Incoming RTPs: UTP, GTP, ATP, CTP • Growing single-stranded RNA released • Adds 30-85 nucleotides/sec (~ 1/10th rate of DNA replication) RNA polymerase reaction

  9. RNA polymerase reaction

  10. Transcription Initiation • Transcription complex assembles at an initiation site (DNA promoterregion) • Short stretch of RNA is synthesized • Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes • Eukaryotic genes each have their own promoter

  11. Transcription of E. coli ribosomal RNA genes

  12. A. Genes have a 5’ 3’ Orientation • Convention for double-stranded DNA:Coding strand (top) is written: 5’ 3’Template strand (bottom) is written: 3’ 5’ • Gene is transcribed from 5’ end to the 3’ end • Templatestrand of DNA is copied from the 3’ end to the 5’ end • Growth of RNA chain proceeds 5’ 3’

  13. Orientation of a gene

  14. Transcription Complex Assembles at a Promoter • Consensus sequences are found upstream from transcription start sites • DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promotersite

  15. Promoter sequences from 10 bacteriophage and bacterial genes

  16. E. coli promoter • (1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp) • (2) -35 region (-35 bp upstream) from start site • Strongpromoters match consensus sequence closely (operons transcribed efficiently) • Weakpromoters match consensus sequences poorly (operons transcribed infrequently)

  17. Initiation of transcription in E. coli(two slides)

  18. Transcription Termination • Only certain regions of DNA are transcribed • Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences

  19. Transcription in Eukaryotes A. Eukaryotic RNA Polymerases • Three different RNA polymerases transcribe nuclear genes • Other RNA polymerases found in mitochondria and chloroplasts • Table 21.4 (next slide) summarizes these RNA polymerases

  20. Eukaryotic Transcription Factors • Same reactions as prokaryotic transcription • More complicated assembly of machinery • Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs)

  21. Transcription of Genes Is Regulated • Expression of housekeepinggenes is constitutive • These genes usually have strongpromoters and are efficiently and continuously transcribed • Housekeeping genes whose products are required at low levels have weak promoters and are infrequently transcribed • Regulatedgenes are expressed at different levels under different conditions

  22. Role of regulatory proteins in transcription initiation • Regulatory proteins bind to specific DNA sequences and control initiation of transcription • Repressors - regulatory proteins that prevent transcription of a negatively regulated gene • Negatively regulated genes can only be transcribed in the absence of the repressor • Activators - regulatory proteins that activate transcription of a positively regulated gene

  23. Inducers and corepressors • Repressors and activators are often allosteric proteins modified by ligand binding • Inducers - ligands that bind to and inactivaterepressors • Corepressors - ligands that bind to and activaterepressors • Four general strategies for regulating transcription

  24. Strategies for regulating transcription initiation by regulatory proteins

  25. Posttranscriptional Modification of RNA • Mature rRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts • In prokaryotes, 1o transcripts often contain several tRNA precursors • Ribonucleases (RNases) cleave the large primary transcripts to their mature lengths

  26. Ribosomal RNA Processing • Ribosomal RNA in all organisms are produced as large primary transcripts that require processing • Processing includes methylation and cleavage by endonucleases • Prokaryotic rRNA primary transcripts ~30S • Contain one copy each: 16S, 23S, 5S rRNA

  27. Eukaryotic mRNA Processing • In prokaryotes the primary mRNA transcript is translated directly • In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm • Eukaryotic mRNA is processed in the nucleus without interfering with translation • In some mRNA, pieces are removed from the middle and the ends joined (splicing)

  28. Eukaryotic mRNA Molecules Have Modified Ends • All eukaryotic mRNA precursors undergo modifications to increase their stability and make them better substrates for translation • Ends are modified so they are no longer susceptible to exonuclease degradation • The 5’ ends are modified before the mRNA precursors are completely synthesized

  29. Guanylate base is methylated at N-7 • 2- Hydroxyl groups of last two riboses may also be methylated

  30. Poly A tails at the 3’ ends of mRNA precursors • Eukaryotic mRNA precursors are also modified at their 3’ ends • A poly A polymerase adds up to 250 adenylate residues to the 3’ end of the mRNA precursor • This poly A tail is progressively shortened by 3’ exonucleases • The poly A tail increases the time required for nucleases to reach the coding region

  31. Some Eukaryotic mRNA Precursors are Spliced • Introns - internal sequences that are removed from the primary RNA transcript • Exons - sequences that are present in the primary transcript and the mature mRNA • Splice sites - junctions of the introns and exons where mRNA precursor is cut and joined

  32. Triose phosphate isomerase gene (nine exons and eight introns)

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