Chapters 13

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Chapters 13 14

• Transcription and Translation
• Note- well cover 12 later!

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Where were going

• Transcription major players, pro and euk
  differences (story)
• Translation major part of the process
• Protein structure NOT covered- but be familiar
  with the terms- primary, secondary, tertiary,
  etc.
• Ch 13s laid out weird- well cover things in
  more of a traditional fashion

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DNA makes RNA makes protein- Central
-------------- Of molecular genetics
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• Prokaryotic Eukaryotic Transcription
  DNA----gtRNA
• I. What making an RNA copy of one strand of
  DNA
• (template, anticoding, antisense strand)
  3'ATCGCCTAGCCGTTAGGG5'
• 5'TAGCGGATCGGCAATCCC3'
• (partner, coding, sense strand)
• transcription
• 5'UAGCGGAUCGGCAAUCCC3'
• II. Importance
• A. Link to Translation
• B. Gene regulation genes are turned on and off
  mainly by transcription.

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• III. Main player(s) RNA polymerases enzymes
  that cause transcription.
• components (prokaryote- eukaryotes MUCH more
  complicated)
• core a,aß,ß Core non-specific binding to DNA
  and transcription of nicked DNA.
• ---------
• a,aß,ß s
• Holoenzyme
• Core s (holoenzyme) specific transcription
  from promoters

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Initiation, Elongation, Termination

• Initiation
• Loose binding to DNA (not at promoter)
• Binding to promoter (closed promoter) helix is
  unwound
• tight binding to promoter (open promoter- the DNA
  is opened!) Note that open is tighter than
  closed!
• First base added, complementary to the anticoding
  strand.
• Many promoters have been sequenced
• -35
  -10 -11
• TTGACaTAtAaTAorG
  upstream
  (purine) downstream

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• Promoter strength strong and weak promoters, up
  and down mutations. Simple control over
  expression.
• Elongation more bases added to the chain, using
  the anticoding strand as the template. Goes _at_ 50
  nucleotides/sec.
• Termination Termination signals- poly U
  hairpin loop
• 5'TACGAATTCGTATTTTTTTTTTT3'
• 3'ATGCTTAAGCATAAAAAAAAA5' transcript forms a
  hairpin
• ----------? ?--------
• 5'UACGAAUUCGUAUUUUUUUUUUU3'
• 3'AUGCUU

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• The hairpin seems to dislodge the RNA pol some
  terminators aided by protein rho.

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Pro-Eu differences

• Polycistronic- ProK
• Monocistronic- Euk

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Replication vs Transcription
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Transcription in Eukaryotes

• Three RNA polymerases
• Transcription factors
• Caps, tails, splicing

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three separate RNA polymerases,

• I- rRNA
• II- mRNA
• III- 5S rRNA, tRNA

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• A LOT more complicated at the start!
• Upstream regulatory sequences- TATA boxes, CAAT
  boxes, enhancers- cis acting elements- need to be
  on the same piece of DNA to have an effect.
• transcription factors LOTS of stuff needs to be
  at the promoter, to get things started! These are
  needed for the proper binding of the RNA
  polymerase to the promoter.
• More cool videos at the DNA replication site on
  transcription.
• http//www.wehi.edu.au/education/wehi-tv/dna/repli
  cation.html

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Splicing MAJOR difference between pro and euk.

• Process snRNPs (snurps) recognize the borders
  of an intron
• Exon / intron
  /Exon
• 5'-------cAG/GUaAGU------YnNAG/G------------------
  3'
• a g
• Y9 pyrimidines (C/U) lariats are formed!
• The process fig 13-13 SNRNPs bind at the 5 and
  branch point, catalyze the splicing, resulting in
  2 exons ligated and a lariat-shaped intron.

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Heres a web site thats got a good illustration
of splicinghttp//www.web-books.com/MoBio/Free/C
h5A4.htm
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Things to Know

• the process of transcription- tell the story of
  initiation, elongation, termination, with the
  players involved
• RNA polymerase (core, holo, sigma factor),
  promoters, (open and closed promoter complexes),
  termination sequences, rho, strong weak
  promoters.
• Pro and euk. Differences monocistronic and
  polycistronic mRNA
• Replication transcription differences 
• cis and trans acting elements- examples.
• Eukaryotic transcription Pol I, II, III
• Cis trans elements, transcription factors,
  enhancers, caps, splicing (tell the story),
  tails, SNRNPs.
• Number of polymerases use of transcription
  factors presence of enhancers requirement for
  transport processing after transcription.   What
  are the products of the splicing reaction?

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Chapter 14- translation
Actually, back to the start of 13- the dogma
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Key points about the code

• read as triplet codons.
• unambiguous each triplet stands for only one AA
• degenerate more than one codon can code for any
  particular AA
• It has start and stop signals, but no internal
  punctuation (commaless).
• (usually) non-overlapping- in theory, you could
  get three proteins (six, if you read it in both
  directions!) out of an RNA sequence, but you
  usually dont- some minor exceptions in bacterial
  viruses.
• code is mostly universal, with a few exceptions.

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Starting met stopping stop codons
Its a one in a million code!
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Wobble (may be optional)

• We dont use 61 different tRNAs
• The third position of the tRNA can wobble,
  allowing for odd base-pairing.
• U pairs with A or G
• I pairs with A, U or G

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Translation and proteins.

• Were going to cover some of the basics of
  translation, and then some of the results, in
  terms of proteins and their modifications.
• The key players the mRNA, the ribosome, and the
  tRNA. Weve just looked at the mRNA, so lets
  look at the other two

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Svedberg unit
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• rRNAs are mostly on one transcript thats
  processed, not spliced.
• They are found in multiple copies, up to 500 in a
  frog, and more in frog eggs- you need multiple
  copies to make all the copies needed in a typical
  cell (10K in a bacterial cell, over 10 million
  in one of your liver cells!). Like the cool
  picture at the start of CH 13- we make massive
  amounts of rRNA! Most of the segments are on a
  single transcript, which are then processed into
  smaller pieces

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• Important parts 1) the anticodon already.
• 2) the 3' end and acceptor stem
• aminoacyl-tRNA synthetase that does this(13-5).
  It costs one ATP (used to charge the COOH, making
  a hi-energy bond), and results in a charged .
• tRNA. There is a single aminoacyl-tRNA
  synthetase for each amino acid. The specificity
  of each is in its ability to recognize certain
  sequences in the acceptor stem. These enzymes
  are important a mutation in one of these would
  cause a global change in the genetic code! It
  would be like a global find and replace in a
  document.

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• Translation Figs 14-6,7. Once again, you have
  initiation, elongation, and termination
• Initiation In prokaryotes, there is a sequence
  at the 5' end that is untranslated, and allows
  binding of the ribosome- ribosome binding site.
• Elongation

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• The Klug/Cummings web site has a good animation
  www.prenhall.com/klug
• Another good animation, on a bunch of stuff  
• http//vcell.ndsu.nodak.edu/animations/home.htm

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Protein function and heredity

• DNA? RNA? Protein? trait (or contribute to the
  trait). Mutations in the DNA can result in
  changes in the protein, often with bad
  consequences.

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Mutations

• Substitutions
• Synonymous- no effect on AA sequence
• Non-synonymous
• Change in the AA (missense)
• Stop codon (nonsense)
• Small deletion/insertion frameshift

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• UNMUTATED-WILD-TYPE
• 5'TTTTATAAATG-CGA-GAC-TAC-GAA-GAA-TTT-CCT-TGC-TTA-
  AAT-CCT-AAC-TGA
  
• MET-ARG-ASP-TYR-GLU-GLU-PHE-P
  RO-CYS- LEU ASN- PRO-ASN-STP
• synonymous substitution (actually very common)-
  CGA- CGG- both arg
• 5'TTTTATAAATG-CGG-GAC-TAC-GAA-GAA-TTT-CCC-TGC-TTA-
  AAT-CCT-AAC-TGA
• MET-ARG-ASP-TYR-GLU-GLU-PHE-PRO-
  CYS- LEU ASN- PRO-ASN-STP

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• Non-synonymous- GAC-gt CAC, Asp-gt His. missense
• 5'TTTTATAAATG-CGA-CAC-TAC-GAA-GAA-TTT-GCC-TGC-TTA-
  AAT-CCT-AAC-TGA
• MET-ARG-HIS- TYR-GLU-GLU-PHE-PRO-CYS-
  LEU ASN- PRO-ASN-STP
• Nonsense CGA-gt TGA, Arg-gt stop.
• 5'TTTTATAAATG-TGA-GAC-TAC-GAA-GAA-TTT-CCT-TGC-TTA-
  AAT-CCT-AAC-TGA
• MET-STP-ASP-TYR-GLU-
  GLU-PHE-PRO-CYS- LEU ASN- PRO-ASN-STP

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• (Added material- not testable at this point)
• Nonsense suppressed by a suppressor tRNA
• ACCtrp
• ACUtrp
• 5'TTTTATAAATG-TGA-GAC-TAC-GAA-UAA-TTT-CCT-TGC-TTA-
  AAT-CCT-AAC-TGA
• MET-TRP-ASP-TYR-GLU-
  GLU-PHE-PRO-CYS- LEU ASN- PRO-ASN-STP