Chapter 12

Lecture Outline

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Chapter 12 Gene Expression at the Molecular Level

•  Overview of Gene Expression

•  Transcription

•  RNA Processing in Eukaryotes

•  Translation and the Genetic Code

•  The Machinery of Translation

•  The Stages of Translation

2

Key Concepts:

Information Flow From Genes to Proteins

Central Concepts

•  One-gene, one-polypeptide hypothesis –  However, not all genes encode for proteins (Regulatory RNA)

•  Central Dogma of Molecular Biology (information flow)

–  DNA to RNA to protein

–  Transcription then translation

Different Types of RNAs

•  RNAs –  mRNA: Carries a copy of the gene sequence from

DNA to the ribosome for translation

–  tRNA: Carries specific amino acid to the ribosome for translation

–  rRNA: Catalyzes peptide bond formation and provides structural framework for the ribsomome

RNAs

Gene Structure

Eukaryotic DNA Transcription: Gene Structure

•  Nucleus separates transcription and translation in eukaryotes

•  Promoters

–  Prokaryotes use one promoter for multiple genes

•  RNA Polymerase can bind by itself

–  Eukaryotes use one promoter for each gene

•  RNA Polymerase requires assistance of other proteins to bind

Eukaryotic DNA Transcription: Gene Structure

•  Exons

–  Protein coding regions

–  In some cases, separate exons encode different protein domains

•  Introns

–  Non-coding regions; could be regulatory

–  Most eukaryotic genes contain and only rarely do prokaryotes have them

•  Terminators –  Stop transcription and RNA polymerase dissociates

RNA Transcription

Figure 14.2 From Gene to Protein

Figure 14.3 RNA Polymerase

Figure 14.4 DNA Is Transcribed to Form RNA (Part 2)

Figure 14.4 DNA Is Transcribed to Form RNA (Part 3)

RNA Processing

Figure 14.10 Processing the Ends of Eukaryotic Pre-mRNA

DNA Transcription: RNA Processing

•  Pre-mRNA – Exons + Introns

•  Mature mRNA – Exons only

RNA Processing: 5’ Cap and Poly A Tail

•  5’ methyl guanosine cap (G cap) –  Added during transcription

–  Chemically modified GTP

–  Facilitates mRNA binding to ribosome and protects from endonuclease digestion

•  Poly A tail –  Added to the 3’ end of mRNA

–  AAUAAA in pre-mRNA after the last codon is a signal to cleave and add a stretch of 100-300 adenine nucleotides

–  Added at the end of transcription

RNA Splicing

Figure 14.7 Transcription of a Eukaryotic Gene (Part 2)

RNA Processing: RNA Splicing

•  Small nuclear ribonucleoprotein particles (snRNPs) –  RNA-protein particles

•  Consensus sequences-Splice sites –  snRNPs binds to consensus sites

•  Splicesome –  snRNPs + other proteins –  Acts to excise intron and join exons

•  Export through nuclear pores –  TAP binds to the 5’ end of mRNA and interacts with the nuclear

pore and other proteins

–  Allows for mRNA export from the nucleus through the nuclear pore

Figure 14.11 The Spliceosome: An RNA Splicing Machine

The Genetic Code

Genetic Code: Characteristics

•  Codon: 3 bases on mRNA

•  Anticodon: 3 bases complementary to the codon on tRNA

•  tRNA carries a specific amino acid

•  4x4x4=64 combinations of codons but the cell typically uses less tRNAs (~40)

Figure 14.6 The Genetic Code

Figure 14.12 Transfer RNA

Wobble Phenomenon

•  Binding specificity tRNA anticodon for 3rd base in the codon sequence of mRNA is not always strictly observed: This phenomenon is called wobble.

•  Each mRNA codon binds to just one tRNA species which carries a specific amino acid

•  But each tRNA could bind to multiple codons due to the wobble position.

Genetic Code: Redundant Not Ambiguous

•  Genetic code is redundant but not ambiguous

•  Redundant: –  More than one codon that specifies a particular amino

acid

•  Not ambiguous: –  Each tRNA binds to only one amino acid

–  Each codon binds to only one type of tRNA/amino acid combination

Protein Translation: RNA to Protein

Figure 14.15 The Initiation of Translation (Part 1)

Figure 14.12 Transfer RNA

Transfer RNA

•  Functions of tRNA

–  “Charged” tRNA is bound to a specific amino acid

–  Associates with mRNA

–  Interacts with ribosomes

•  Structural elements of tRNA

–  Secondary structure; internal hydrogen bonding of tRNA sequence

–  3’ end-Amino acid attachment site (covalently bound)

–  Unique anticodon-Midpoint of tRNA binds complementary sequence in mRNA

Ribosomes

•  Peptide synthesis factories

•  Two subunits: Large and small subunits –  Eukaryotes: 3 rRNA + 49 proteins (Large subunit);

1 rRNA + 33 proteins (Small)

–  Non-covalent interactions hold subunits together

–  Prokaryotes similar, just smaller

Translation at a glance

•  Large subunit

–  A (Amino acid) site: Charged tRNA binding site

–  P (polypeptide) site: Where tRNA adds its amino acid to the growing polypeptide chain

–  E (exit) site: uncharged tRNA leaves from here

•  Fidelity function: rRNA from small subunit validates the three base-pair match

–  Without appropriate hydrogen bonding the tRNA is released

Figure 14.14 Ribosome Structure

Figure 14.15 The Initiation of Translation (Part 2)

Initiation Complex

Initiation Factors

Figure 14.16 The Elongation of Translation (Part 1)

Peptidyl-transferase activity

Figure 14.16 The Elongation of Translation (Part 2)

Figure 14.16 The Elongation of Translation (Part 3)

Figure 14.17 The Termination of Translation (Part 1)

Figure 14.17 The Termination of Translation (Part 2)

Figure 14.17 The Termination of Translation (Part 3)

•  Reading Frame is the codon composition in a mRNA sequence.

•  This is what the ribosome will “read” for protein synthesis during translation.

Figure 14.18 A Polysome

Figure 14.13 Charging a tRNA Molecule with Aminoacyl tRNA Synthase

Charging tRNAs

•  Catalyzed by aminoacyl-tRNA synthases –  Three part active site (amino acid, ATP, tRNA)

•  Very low error rate of: –  Enzyme recognition of specific tRNA

–  Enzyme recognition of specific amino acid

•  “Second Genetic Code”-tRNA charging