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Chapter 15: Genes and How They Work

Chapter Outline

Chapter 15: Genes and How They Work

 

15.0 Introduction
  1. Hereditary Information Directs Building of a Whole Organism

    1. Information Contained in DNA fig 15.1

    2. Proteins Are Tools of Heredity

15.1 The central dogma traces the flow of gene encoded information

  1. Cells Use RNA to Make Protein

    1. Determine Location of Protein Production

      1. Place cells in medium with radioactively labeled amino acids

        1. Cells take up amino acids to construct polypeptide chains

        2. Label first appears in cytoplasm on ribosomes fig 15.2

      2. Structure of ribosomes

        1. Complex molecules composed of several RNA molecules and proteins

        2. RNA similar in structure to DNA fig 15.3

    2. Kinds of RNA

      1. Ribosomal RNA (rRNA)

        1. With proteins, make up the ribosomes

        2. Site of polypeptide synthesis

      2. Transfer RNA (tRNA)

        1. Transport amino acid molecules to ribosome

        2. Position amino acid along growing polypeptide chain

        3. Smaller in size than rRNA, 45 different kinds in human cells

      3. ger RNA (mRNA)

        1. Long strand of RNA copied from DNA

        2. Passes from nucleus to cytoplasm

        3. Conveys information from chromosomes to ribosomes

    3. The Central Dogma fig 15.4

      1. Information passes from genes (DNA) to RNA copy of gene

      2. RNA copy directs assembly of amino acid chain

      3. DNA ® RNA ® protein

    4. Transcription: An Overview

      1. First step is production of mRNA copy of DNA gene

        1. mRNA formed on DNA template

        2. DNA transcribed into RNA sequence

      2. Initiated by RNA polymerase enzyme

        1. Binds to promotor at beginning of a gene

        2. mRNA complementary to DNA assembled

          1. Adenine and thymine pair

          2. Guanine and cytosine pair

          3. New RNA strand contains uracil not thymine

        3. At stop signal polymerase disengages, mRNA is released

        4. mRNA made is complementary RNA transcript of DNA information

    5. Translation: An Overview

      1. Information in mRNA directs synthesis of polypeptide on ribosome

      2. mRNA information translated into amino acid sequence of polypeptide

      3. Initiated by rRNA molecule of ribosome that binds to mRNA "start"

        1. Ribosome moves along mRNA chain in three nucleotide groups

        2. Disengages at "stop" signal, polypeptide is released

      4. Transcription plus translation result in gene expression

15.2 Genes encode information in three-nucleotide codewords

  1. The Genetic Code

    1. Proving Codewords Have Three Letters

      1. Codons are blocks of information corresponding to amino acids

      2. Postulated code was three nucleotides long

        1. Two nucleotide block would code for only 16 amino acids

        2. 20 known amino acids

        3. Three nucleotide block would code for 64 amino acids

      3. Questioned whether code was simple or punctuated

        1. In simple code, each nucleotide is part of a codon

        2. Punctuated code has spacer nucleotide between codons

      4. Experimental process involved altering reading frame fig 15.5

        1. Chemically deleted one, two or three nucleotides from viral DNA

        2. Allowed transcription to proceed

        3. Change of less than three caused nonsense reading

        4. Change in three nucleotides restored reading frame

        5. Same results when one, two or three nucleotides added

      5. Concluded code was continuous triplet code, not punctuated with spacers

    2. Breaking the Genetic Code

      1. Nierenberg produced phenylalanine from polyU mRNA

        1. Added artificial RNA to cell-free RNA and protein

        2. Concluded UUU coded for phenylalanine

      2. Nierenberg and Leder developed triplet binding assay

        1. Determined which radioactive amino acid would bind to each triplet codon

        2. 47 of 64 codons gave unambiguous results

        3. Other 17 determined by making artificial mRNA, examining resulting polypeptide

      3. All 64 sequences tested and genetic code determined tbl 15.1

    3. The Code Is Practically Universal

      1. Most of the code is same in all organisms

        1. Strongest evidence that all living organisms share common heritage

        2. Genes transcribed by one organism can be translated by another

        3. Genes can be transferred from one organism to another

      2. Universality is central to advances of genetic engineering

        1. Insulin made by human genes inserted into bacteria

        2. Bacteria are factories producing large quantities of the enzyme

    4. But Not Quite

      1. Genetic code of mammalian mitochondria not same

        1. UGA in reads for tryptophan not "stop" as in universal code

        2. AUA reads as methionine not isoleucine

        3. AGA and AGG read as "stop" not arginine

      2. Other minor differences found in chloroplasts and some single-celled ciliates

      3. After endosymbiosis some mitochondria and chloroplasts began to read code differently

        1. Changes in stop signal would normally be lethal

        2. Explanation not evident

15.3 Genes are first transcribed, then translated

  1. Transcription

    1. First Step in Transcription Requires an Important Enzyme fig 15.6

    2. RNA Polymerase

      1. Structure of bacterial enzyme as a model

        1. Large, complex molecule composed of five subunits

        2. Two a subunits bind regulatory proteins

        3. One b' subunit binds to DNA template

        4. One b subunit binds RNA nucleotide subunits

        5. One s subunit recognizes promotor, initiates synthesis

      2. Only one strand is synthesized, the template strand (also + or sense strand)

        1. Transcript is complementary to original DNA

        2. Untranscribed strand is coding strand (also - or antisense strand)

        3. Coding strand has same sequence as mRNA with T for U exchange

      3. In all organisms polymerase adds ribonucleotides only to 3' end of chain

      4. No primer needed, proceeds in 5' ® 3' direction

      5. Differences between bacterial and eukaryotic transcription

        1. Bacteria have only one RNA polymerase enzyme

        2. Eukaryotes have three RNA polymerases

          1. RNA polymerase I makes rRNA in nucleolus

          2. RNA polymerase II makes mRNA

          3. RNA polymerase III makes tRNA

    3. Promotor Sites

      1. Site on DNA where RNA polymerase binds at the start of transcription

      2. Bacterial promotor site is about 60 base pairs long, not transcribed itself

      3. Almost all bacteria have two same six-base sequences in promotor site

        1. TTGACA sequence, -35 sequence, located 35 nucleotides upstream of start

        2. TATAAT sequence, -10 sequence, 10 nucleotides upstream of start sequence

      4. Eukaryotes have sequence similar to bacterial -10 sequence

        1. TATAAA sequence, called TATA box

        2. Located at -25, further from start site than bacterial -10

      5. Promotors differ in efficiency

        1. Strong promotors cause frequent initiations of transcription (every 2 seconds)

        2. Weak promotors transcribe less frequently (every ten minutes)

        3. Strong promotors have unaltered -35 and -10 sites

        4. Weak promotors may have substitutions at these sites

    4. Initiation

      1. First step is binding of RNA polymerase to promotor

      2. In bacteria, s (sigma) subunit of RNA polymerase recognizes -10 region

        1. Subunit can detect region without unwinding the DNA

        2. Binds RNA polymerase to site

      3. In eukaryotes, -25 sequence serves similar function

        1. Is binding site for a key protein factor

        2. Other eukaryotic factors bind forming a complicated transcription complex

      4. When bound to promotor RNA polymerase begins to unwind DNA helix

        1. Bacterial polymerase unwinds 17-base-pair segment

        2. Sets stage for assembly of RNA chain

    5. Elongation

      1. Transcription of RNA chain starts with ATP or GTP

        1. Forms 5' end of chain, grows in 5' ® 3' direction

        2. Primer not required as it is in DNA synthesis

      2. Region of transcription called the transcription bubble

        1. Contains a locally unwound "bubble" of DNA fig 15.7

        2. First 12 bases of RNA form temporary helix with DNA template strand

        3. Stabilizes positioning of 3' end of RNA for interaction with new nucleotide

        4. RNA-DNA hybrid helix rotates each time a nucleotide is added

        5. Keeps 3' end of RNA at catalytic site

      3. Transcription bubble moves down DNA at constant rate

        1. Speed of 50 nucleotides per second

        2. Growing RNA strand protrudes from bubble

        3. Transcribed DNA rewinds after transcription

      4. RNA has no proofreading capacity

        1. More copying errors in transcription than in DNA synthesis

        2. Mistakes not transmitted to progeny

        3. Many mRNA copies made, a few faulty copies are not harmful

    6. Termination

      1. Stop sequences located at end of gene

        1. Cause cessation of formation of phosphodiester bonds

        2. Dissociates RNA-DNA hybrid within transcription bubble

        3. RNA polymerase releases DNA

        4. DNA within bubble rewinds

      2. Examples of stop signals

        1. Simplest is series o f GC pairs followed by series of AT pairs

        2. Forms GC hairpin, followed by series of U nucleotides fig 15.8

      3. How hairpin structure causes termination

        1. RNA polymerase pauses after it synthesizes the hairpin

        2. Polymerase thus positioned over U series

        3. Bond between RNA U and DNA A is weak, can't hold hybrid together during pause

        4. RNA strand dissociates from DNA template, transcription stops

        5. In particular genes, variety of protein factors may assist

    7. Posttranscriptional Modifications

      1. Eukaryote mRNA transcripts must travel out of the nucleus, into the cytoplasm

      2. Eukaryote mRNA transcripts are modified to aid in the journey

      3. 5' cap modification

        1. Transcripts usually begin with A or G, modified in eukaryotes

        2. GTP added backwards, forms unique 5'-5' linkage

        3. Protects 5' end of RNA transcript from degradation by nucleases and phosphotases

      4. 3' polyA tail modification

        1. Forms after eukaryotic transcript is released from transcription bubble

        2. 3' end cleaved at site with AAUAAA sequence

        3. PolyA polymerase adds 250 A ribonucleotides to 3' end of transcript

        4. Long string of As protects transcript from nuclease degradation

        5. Makes transcript a better template for protein synthesis

  2. Translation

    1. First Events in Translation

      1. Initial portion of mRNA binds to rRNA in ribosome

      2. Single mRNA codon exposed at polypeptide-making site

      3. tRNA with complementary anticodon binds to mRNA fig 15.9

        1. Anticodon three nucleotides long

        2. Each tRNA specific for an amino acid

      4. Successive codons exposed, series of tRNAs bind to exposed codons

      5. Successive amino acids added to growing string of polypeptides fig 15.10

      6. Rationale for more codons than there are tRNAs

        1. 64 codons, 45 kinds of tRNA

        2. Third base pair in codon allows for "wobble"

        3. Some tRNAs recognize more than one codon

      7. Proper pairing of codon and tRNA depends on activating enzymes

    2. Activating Enzymes

      1. One aminoacyl-tRNA synthetase exists for each of 20 common amino acid fig 15.11

      2. Activating enzymes specify amino acid to be added to tRNA

        1. Each corresponds to anticodon sequence and amino acid

        2. Each recognizes different identities and numbers of tRNAs

        3. Each still codes for only one amino acid tbl 15.1

    3. "Start" and "Stop" Signals

      1. Special, non-amino acid associated codons

        1. Nonsense codons are stop signals: UAA, UAG, UGA

        2. AUG is the start signal

      2. Summary of translation fig 15.12

  3. A Closer Look at the Mechanism of Protein Synthesis

    1. Initiation

      1. In prokaryotes synthesis begins with initiation complex

      2. tRNA with N-formylmethionine (fMet-tRNA) binds to small ribosomal subunit

      3. Three special sites exist on ribosome surface

        1. Peptidyl, P site, is where peptide bonds will form

        2. Aminoacyl. A site is where successive amino acid-tRNAs will bind

        3. Exit, E site, is where empty tRNAs exit the ribosome fig 15.13

      4. Initiation factors position fMet-tRNA at p (peptidyl) site on ribosomal surface

        1. Initiation complex, guided by another initiation factor, binds to mRNA at AUG

        2. Positioning critical to reading frame of mRNA

        3. Complex must bind to beginning so whole transcribed gene is translated

      5. Bacterial mRNA start with leader sequence

        1. Complementary to one rRNA on ribosome

        2. Allows base pairs to form between mRNA and rRNA

      6. Bacteria and eukaryotes differ in number of genes per mRNA transcript

        1. Several genes in one bacterial transcript (polycistronic)

        2. One gene per eukaryotic transcript (monocistronic)

      7. Differences in eukaryotic initiation

        1. Initiating amino acid is methionine not N-formylmethionine

        2. Initiation complex is more complicated

    2. Elongation

      1. Large ribosome subunit binds, exposes codon adjacent to initiating AUG

        1. Elongation factors assist in binding tRNA to exposed mRNA codon on A site

        2. Amino acid on tRNA adjacent to initial methionine fig 15.14

      2. The two amino acids chemically react with one another

        1. Catalyzed by peptidyl transferase

        2. Methionine released from its tRNA

        3. Attached by peptide bond to adjacent amino acid

      3. Translocation occurs fig 15.15

        1. Ribosome moves along mRNA to next codon

        2. Relocates previous tRNA to E site

        3. Repositions tRNA with growing polypeptide to P site

        4. Exposes next codon at A site for incoming tRNA

        5. tRNA recognizing next codon binds to A site

      4. Process continues repeatedly from step B.2.

    3. Termination

      1. Elongation continues till chain-terminating nonsense codon appears fig 15.16

      2. No tRNA binds to nonsense codons

      3. Recognized by special release factors

15.4 Eukaryotic gene transcripts are edited

  1. The Discovery of Introns

    1. Primary Difference Between Prokaryote and Eukaryote Protein Synthesis

      1. Eukaryotic genes much longer than necessary

        1. Stretches called introns not translated

        2. Do not correspond to any portion of a polypeptide

        3. Exons are remaining, polypeptide specifying portions

      2. In bacteria virtual all transcribed nucleotides are necessary to produce protein

      3. In eukaryotes, proteins coded for by RNA segments from several locations

        1. Primary RNA transcript (primary transcript) contains all information

        2. RNA segments spliced together to form active mRNA transcript to be translated

      4. Experimental procedure that determined splicing process

        1. Isolation and purification of transcribed mRNA

        2. Synthesis of complementary strands with reverse transcriptase

          1. DNA molecules called copy DNA or cDNA

          2. Has same nucleotide sequence as original DNA of gene

        3. Portion of nuclear DNA containing gene is isolated and cloned

        4. Single stranded forms of cDNA and cloned DNA mixed, hybridize

      5. Experimental results

        1. DNA was not single duplex, but also formed unpaired loops

        2. Concluded lengths of DNA removed from transcript before translation

        3. Removed sequences called introns

        4. Remaining sequences called exons fig 15.17

        5. Introns, being excised, do not affect structure of protein encoded by "their" gene

    2. RNA Splicing

      1. Primary transcript contains sequences complementary to the entire gene

      2. Introns cut out during RNA processing (RNA splicing)

        1. Sequences not translated

        2. Remaining exon sequences are spliced together forming final mRNA

        3. In typical human gene introns may be 10 to 30 times larger than exons

        4. Hemoglobin primary mRNA transcript contains 1356 nucleotides

        5. Only 432 nucleotides code for 144 amino acids in hemoglobin

      3. Summary of eukaryotic protein synthesis fig 15.18

  2. Differences Between Bacterial and Eukaryotic Protein Synthesis

    1. Six Primary Differences

      1. Introns

        1. Most eukaryotic genes have introns

        2. A few Archaebacteria have introns

        3. Other prokaryotic genes lack introns fig 15.19

      2. Number of gene transcripts in one mRNA molecule

        1. Bacteria mRNA molecules may contain transcripts of several genes

        2. Bacteria coordinate regulation of gene function by putting them on same mRNA

        3. Eukaryotic mRNA usually contains transcripts of only one gene

        4. Eukaryotic gene regulation achieved in other ways

      3. Separation of transcription and translation

        1. Eukaryotes mRNA must pass through nuclear membrane before translation

        2. Bacterial mRNA begin translation before transcription is complete

      4. Initiation of translation

        1. Bacteria translation begins at AUG preceded by special nucleotide sequence

        2. Eukaryote mRNA modified with 5' cap that initiates translation

      5. Modification of mRNA for translation

        1. Bacteria mRNA translated directly as transcribed

        2. Eukaryotic mRNA add polyA tail, protects molecule from degradation

      6. Ribosome size: Eukaryote ribosomes larger than bacterial ribosomes

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