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Last revised: Friday, October 15, 1999
Reading: Ch. 17 in text

Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!

Copyright 1999. Thomas M. Terry

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The Expression of Genetic Information: an Overview

• All cells contain enormous amounts of DNA. Even the smallest known cell, a bacterium named Mycoplasma genitalium, contains nearly 500,000 base pairs (500 kilo base pairs, or 500 kbp).
• What do cells do with all this information? In bacteria (the simplest cells), DNA serves two major functions:
  1. encoding structural information that can be converted into RNA and (usually) thence into protein sequence.
  2. encoding regulatory signals that allow certain proteins to decide where to begin or terminate reading DNA
• In eukaryotic cells, where DNA also has to undergo condensation into chromosomes and mitotic or meiotic events, DNA also contains specialized sequences used for centromeres, telomeres, and other functions.
• The "central dogma" of molecular biology: information flows from nucleic acids to proteins, not the reverse:

  DNA ----> RNA ----> polypeptides (proteins)
  View graphic summary of information flow

• Transcription = the process of making RNA from DNA templates
• Translation = the process of making polypeptides
• View example of how information is coded from DNA to peptide

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Transcription: the Synthesis of RNA

Work through From Gene to Protein: Transcription at The Biology Place..

Structure of RNA

• Components: Ribose, Phosphate, A, C, G & U.
• View RNA components (from The Biology Place)
• U has same base pairing properties as T (forms U=A base pairs)
• RNA is not double stranded (except in some viruses)
• RNA can have extensive "hairpin" loops
• RNA can have modified bases (after transcription) -- find unusual bases such as inosine, pseudouridine. These still have base pairing properties, and contribute to stability of molecule,
• Messenger RNA has half-life of only 3 minutes in bacteria, so cell must constantly make new messages to make new proteins-- allows rapid adaptation to new environments.
• Eucaryotic RNA is controlled very differently. RNA synthesized in nucleus, modified (to be discussed), and exported to cytoplasm before it is translated.

Types of RNA

• messenger RNA -- carries codons to RNA
• ribosomal RNA -- part of ribosome structure, catalyzes peptide bond formation
• transfer RNA -- set of small RNAs, transport amino acids to ribosome for incorporation into growing polypeptides

Transcription Process

RNA polymerase enzyme

• View animation of beginning of transcription (from The Biology Place.).
• opens up DNA helix for short stretch (~ 15 base pairs)
• selects one of two strands as template strand
• RNA synthesized in 5' to 3' direction
• RNA synthesis begins at promoters: sites on DNA that are recognized as "start" signals for RNA synthesis.
• Terminators: regions where RNA synthesis stops, RNA is released from DNA.
• View animation of transcription from start to finish (from The Biology Place.).

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Translation: the Synthesis of Proteins

• View animation of translation process from Univ. of Virginia
• Work through The Translation process at The Biology Place

role of mRNA

• carries codons (3-nucleotide sequences) arranged in linear fashion that code for amino acids
• also carries signals needed to tell how to recognize ribosomes, start and stop signals for decoding protein
• leader sequence on small ribosome subunit binds to complementary sequence on mRNA, allows initial formation of RNA-ribosome complex.
• View RNA

role of ribosome

• acts as a "decoding box" or "tape player" for the information in mRNA
• two parts: a small subunit and a large subunit. These are separated except when attached to m-RNA
• ribosomes contain a set of ribosomal proteins and several types of ribosomal RNA (r-RNA)
• ribosomes catalyze the formation of peptide bonds; intially thought to be due to protein activity (enzyme), but now known to be due to RNA catalytic activity (ribozyme)
• View ribosome

role of tRNA

• structure: 4 loops, anticodon, AA binding site
• View model of tRNA
• ~ 60 types in bacteria (>100 in mammals)
• only 73-93 nucleotides long
• some bases modified after transcription; form "funny bases" like pseudouridine.
• extensive hairpin loops
• anticodon site: recognizes codon on mRNA
• Activation of tRNA: adding amino acids
  • requires special enzyme: AA-tRNA activating enzymes
  • ATP required, forms AA-AMP + PP, then AA-tRNA + AMP
  • View animation of activation process

Initiation of Translation

• small ribosomal subunit initiates binding to mRNA
• locates 5' end of mRNA
• small subunit ribosome finds first AUG codon = start codon
• large ribosome binds
• tRNA carries the amino acid methione to first position
• view animation

Elongation of Translation

• 2 adjacent sites on ribosome: P (Peptide) and A (Amino Acid) site
• A site accepts a new tRNA-AA
• P site holds existing chain
• peptide transferred from P site tRNA to A-site AA
• enzyme activity is in ribosomal RNA (ribozyme)
• View animation
• also required: Energy (GTP) and elongation factors

Termination of Translation

• reach a "stop codon" UAG, UAA, or UGA
• View animation
• no t-RNAs bind
• instead, specific release factors required
• Net cost of translation: 4 phosphate bonds/amino acid added!

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The Genetic Code

• View Genetic Code table
• 64 possible codons (3-letter nucleotide sequences): AAA, AAC, etc.
• 60 codons serve as straightforward signals for amino acids
• Some amino acids coded by a single codon
• Some amino acids coded by as many as six different codons (redundancy)
• One codon serves as universal "start": AUG (memnonic: "A, you go!")
• Three codons serve as "stop" signals: UAG, UAA, UGA (memnonic: "You are gone, You all away, You go away")

Universality

• originally thought all organisms use identical codons
• But mitochondria of eukaryotes (except plants) use slightly different assignments for a few codons. Examples:
  1. UGA = stop (univ), or amino acid Tryptophan (mitochondria from yeast, protozoans, mammals)
  2. AUA = Isoleucine (univ), or Methinonine (mitochondria from yeast, protozoans, mammals)

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Gene organization in Eukaryotes

The coding potential of human DNA

• human DNA contains 6 x 10⁹ base pairs/cell = 6,000,000 kb pairs
• compare to 4700 kb pairs/E. coli, a very sophisticated bacterium. Human DNA is more than 1000x bigger!
• If all human DNA coded for proteins, would have enough for roughly 5 million different proteins
• But currently only know ~ 3000 human proteins, and estimates as to how many we truly have range from 10,000 to 100,000
• In fact, less than 5% of human DNA codes for protein!
• What does the rest of the DNA do?

Functions of human DNA

• Coding for proteins. Eukaryotic genes are organized in peculiar fashion:
  1. Exons: (short for "expressed") -- regions of DNA that code for amino acids.
  2. Introns: (short for "intervening" or "interrupting") -- regions of DNA inside a gene, located in between exon regions, but not coding amino acids
  3. When RNA is transcribed from a gene, it initally contains both introns and exons, and cannot be called "messenger RNA" yet because the message is interrupted. Introns must be removed by "cut-and-paste", called RNA splicing.
     • View animation of RNA splicing in eukaryote (from The Biology Place.).
  4. snRNPs ("snurps") = small ribonucleoprotein particles, found in nucleus. Composed of RNA and a few proteins. snRNPs associate to form a Spliceosome, which locates the junction of intron and exon, specifically cuts at this junction, and joins the cut ends of exons to form messenger RNA.
  5. Ribozymes: the enzymatic activity of spliceosomes was initially thought to be in the protein. However, now known to be on RNA; first example of catalytic RNA (called ribozyme for as opposed to enzyme, which is protein).
  6. Note: almost all genes in eukaryotes contain intron/exon organization. In some cases, amount of intron can be much larger than amount of exon DNA.
  7. Evolutionary importance of introns: since many proteins consist of several domains with different functions,
• Multigene Families: some genes are represented by more than one copy, typically for products needed in large quantity by cell.
  1. Example 1: ribosomal genes (for ribosomal RNA). Copies of the same gene are clustered together in enormous number (hundreds of thousands of identical gene copies).
  2. Example 2: histone genes (for proteins that bind to DNA to make chromatin). Family of histone proteins is represented many times.
• Pseudogenes: examples of multigene families where some copies of the gene have mutated to the point where they no longer function at all in the cell.
  1. Example: globin gene family. In humans, find several slightly different globin genes that produce the hemoglobin molecules needed by fetus, embryo, and adult. But also find a cluster of genes nearly identical in base sequence, but never expressed in the life of a human.
  2. Explanation: at some time in evolutionary past, globin genes were duplicated (by gene transposition). One cluster retained the job of making functional hemoglobin. The other cluster mutated so that promoter site no longer could be recognized by RNA polymerase. Result = this gene cluster now serves no purpose, cannot make any RNA or protein, but provides evidence of an evolutionary past. Called a pseudogene because it looks like a gene, but doesn't function.
• Repetitive sequence DNA. Some regions of DNA contain short sequences repeated many thousands of times = "tandem repeats". No coding function at all.
  1. Example 1: "satellite" DNA. Sequence such as ACAAACT repeated again and again (producing ...ACAAACTACAAACTACAAACTACAAACTACAAACTACAAACT...). These regions appear to be located where the centromere forms, so this sequence must have mechanical properties that allow recognition by kinetochore and mitotic spindle.
  2. Example 2: "telomeric" DNA. Sequences such as TTAGGG repeated over and over, 250-1500 times. Found at the ends of linear chromosomes (telomeres) where RNA primase (needed to prime the synthesis of new DNA) cannot work on lagging strand. Telomeric DNA acts like a "cap" on the end of the chromosome. If didn't have this, then DNA would lose a bit every replication, chromosome would gradually get shorter.

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