Transcription: DNA?RNA 11/17 and 11/19

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Transcription DNA?RNA11/17 and 11/19

• RNA vs DNA how do their structures differ?
• DNA Double helix and antiparallel H-bonds
• What are codons and why are they important?
• What are the steps to transcription in
  prokaryotes?
• What are the steps to transcription in
  eukaryotes?
• What are the RNApolymerase types?
• What are transcription factors?
• Introns and Exons RNA processing
• Where does tRNA and rRNA come from?

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Suggestions for term paper drafts

• Get another student or four to proof-read again
  just to double check your changes.
• If you like I can put papers in my box at
  officeyou can put one in to be edited and take
  one out to edit do you want to voluntarily make
  this possible?
• Be sure each item in text relates specifically to
  your title, feel free to delete materials. Over
  all length of final draft text is reduced to 3
  page minimum to help you, although making it
  longer than 3 pages is fine.
• Abstract vs. Conclusion Make them different?this
  is a tough one to do.
• Brutal detail but short (no fluff) vs. Nice easy
  to read list/review of key points a possible
  sentence or two about what the future of this
  sort of research will be/
• Introduction Tell the reader what topics you
  will discuss/teach and perhaps provide a bit of
  background info if really needed/
• Try to have a minimum of two different references
  sources cited in each paragraph. Try for at least
  3 sentences or 4 lines of text in each paragraph.
• Write to remove fluff (it hurts to take things
  out that represent neat info) that is not VERY
  specific to your paper title, if it does not
  relate to the title it probably should be removed
• With regards to content in paper you need to look
  for repeated items/info and please remember
  REPEAT ? DELETE or rephrase/reword

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Bases of DNA and RNA

• Base Choices for DNA Adenine, Thymine, Cytosine
  and Guanine
• Base Choices for RNA Adenine, Uracil, Cytosine
  and Guanine
• Base Pairing in DNA duplex AT and GC
• Base Coding from DNA to RNA TRANSCRIPTION
• Transcription is performed by RNA Polymerase
  (RNAP)
• DNA bases ? transcribed into RNA bases
• Ad--gtUr
• Td--gtAr
• Gd--gtCr
• Cd--gtGr
• mRNA sequence (codons) are TRANSLATED into an
  amino acids linked by peptide bonds.

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Nucleoside consist of a ribose (3 OH-sugar),
base (A,T, G,C, or U) and a triphosphate. Link
the phosphate to ribose with a phosphoester bond
(cut off two phosphates) and your have RNA (R-OH)
or DNA (R-H).
Becker_6e_IRCD_Chapter_3
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Nucleotide ? remove PHOSPHATES? left with a
Nucleoside
Becker_6e_IRCD_Chapter_3
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Hydrogen Bonds between two antiparallel
nucleotide chains create the base pairs that
stabilize your DNA (thats why RNA is unstable)
Becker_6e_IRCD_Chapter_3
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TRANSLATION The key to making a protein is to
find the three ribonucleic acids on mRNA that
make Methionine (met) and to move 1 codon
(triplet) and an additional amino acid/codon in
the 3 direction until a stop codon is reached.
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Prokaryotic Transcription (RNA formation) is a
four step cycle

• 1) Template Binding template is recognized and
  bound by RNAP at a special promotor sequence
• 2) Chain Initiation a dinucleotide is formed
  from the template from ATP and an XTP
• (XAdenine, Guanine, Uracil, Cytosine plus
  ribose-5 phosphate) (no thymine in RNA it is
  in DNA only)
• After initiation the Sigma factor falls of RNAP
• 3) Chain Elongation new bases are added to the
  nascent 3 OH end of the mRNA.
• Initiating factor gets bumped off after the first
  10 bases
• 4) Chain Termination/Release RNAP termination
  factor recognizes the termination sequence or a
  special RNA hairpin loop forms that lets RNAP
  fall off and mRNA is released
• Many RNAs can be simultaneously produced from a
  single DNA strand.
• Each strand of DNA can be used to make a unique
  mRNA depending on the reading frame used to make
  initiate the RNA

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Overview of Four Step Transcription Process
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Many mRNAs can be made at the same time from one
DNA.The elongating chains below are mRNAs being
transcribed.
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RNAP must identify promotors -35 and -10 bases
from where initiation begins (1)! The -10
region is called a TATAAT or Pribnow Box on the
5?3) coding strand 3ATTCA5 template
strand. This template strand becomes 5UAAGU3
on mRNA.
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The DNA duplex is unwound by RNAP to create
access to the template strand for transcription
into a complementary RNA sequence. NTPs are ONLY
added (elongation) to the RNA 3 end
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Termination occurs when a termination sequence of
DNA is observed by the Rho factor of RNAP or when
the RNA produced forms special complementary
double stranded RNA sequences that pull RNAP off
of the DNA. Normally RNA does NOT form a double
stranded structure for large lengths/durations.
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Eukaryotic Transcription (DNA? RNA) is more
complex, slower and more flexible than in
Prokaryotes

• Gross differences in prokaryotic and eukaryotic
  transcription.
• 5 Eukaryotic RNA Polymerase subtypes
• Core promoters on eukaryotic DNA
• Transcription factors are required for
  RNAPolymerase-DNA binding/transcription
• Eukaryotic up-regulation and down-regulation of
  RNA production
• Termination of RNA polymerase/transcription
• Relative RNA content in eukaryotic cells
• rRNA production by Polymerase I and processing
• mRNA production by RNApolymerase II and
  processing
• tRNA production by RNApolymerase III and
  processing
• Removal of introns from pre-mRNA in the nucleus
• Function of Spiceosomes

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There are 6 major differences between eukaryotes
and prokaryotes in-terms of how they approach
transcription

• 1) E.T. have 5 different RNAPs, not 1
• 3 in nucleus 1 in mitochondria 1 in
  chloroplast
• 2) E.T. promoters are much more variable
• Variable with respect to identity and location
• 3) E.T. factors (TFs) bind DNA/initiate RNAP
• Allows fine-tuning of RNA production
• 4) E.T. often have additional proteins that
  bind/modify TFs
• 5) E.T. can cleave pre-RNA prior to final product
  formation
• 6) E.T. can modify nucleotides of pre-RNA
• Generally
• Prokaryotes only make proteins when absolutely
  needed
• Eukaryotes can make a bit more/less to fine tune
  production to need

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Relative to prokaryotes, eukaryotes have much
greater flexibility in terms of their ability to
modify protein production. This comes at the
cost of greater complexity and time needed to
make a protein.
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Eukaryotes have 5 different RNA polymerasesEach
subtypes makes a specific type of RNA

• 1) RNAP I makes rRNA in the nucleolus (darkest
  part)
• 2) RNAP II makes mRNA and snRNA in nucleoplasm
• 3) RNAP III makes rRNA and tRNA in nucleoplasm
• RNAPolymerases are massive complexes 500,000mw
• Similar to Prokaryotic RNAPolymerases
• 4) Mitochondria has mRNAP
• 5) Chloroplast has cRNAP
• How much of each type of RNA are contained in a
  cell? rRNA about 75
• tRNA about 15
• mRNA less than 10

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Transcription in Eukaryotes

• A core promoter must be present near where RNA
  transcription is to begin!
• Many types of promoter exist
• Each RNAP type (I, II, III) looks for its unique
  promotor and synthesizes its unique RNA product
  type
• Promoter sequence on DNA may be upstream or
  downstream of initiation site
• Upstream before initiation site ( 5 end of 1
  on template DNA)
• Downstream after initiation site (3 end of 1
  on template DNA)

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RNAP II makes mRNA! Several sequence patterns
help make RNAP binding to mRNA possible
TATA-box, GC-box and the CAAT-box.

• TATA Box is at -25 , not -10 as in prokaryotes
• GC- and CAAT-boxes are variable located -40 to
  -100 bp upstream of the 1 site modify
  TATA-box/RNAP affinity
• They can be on the template OR coding DNA strands
• They improve affinity of RNAP for TATA box
• Classic eukaryotic promotors on mRNA

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Transcription Factor II core proteins let RNAPII
bind DNA and begin transcription. Special TFs
for RNAPI/RNAPIII also exist!

• 1) TFIID- binds TATA DNA (often called a TBP)
• 2) A and B TFIIs bind/modify TFIID
• 3) RNAP II(TFIIF) can now bind at TATA box near
  1
• 4) TFII-E binds and forms pre-initiation
  complex
• 5) Binding TFII-H permits DNA unwinding
  (helicase) and phosphorylation of RNAPII
  (activation)
• 6) Initiation occurs TFIIA/D released and
  transcription begins
• Dont forget that upstream histones and
  chromatins on DNA must also be removed prior to
  DNA unwinding/transcription.
• 7) Termination of mRNA transcription by RNAPII
  occurs when a termination factor recognizes AAUAA
  in the RNA
• Steps 1D?2A/B?4RNAPII?5E/H?A/D Release?
• Initiation complete and Transcription Begins!

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• Transcriptional Regulation Eukaryotes have the
  unique ability to enhance/suppress transcription
  of a gene to better match needs for proteins that
  are non-constitutive in nature.
• This is how steroid hormones work in cells to
  modify basal production so expensive protein
  production is fine tuned to need.
• Enhancers/Silencers are generally 100s to 1000s
  of nucleotides away from the TATA box

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What does the finished product look like when
transcription is moving ahead?
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Pre-mRNA is processed in several ways including
1) Capping of the 5 end with methyl-groups
immediately after transcription2) Adding a poly
A tail 3) Removal of intron sequences.
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Introns located inside the pre-mRNA represent
junk that must be properly removed by
spliceosomes prior to departure from the nucleus.
Spliceosomes recognize/cut consensus sequences
OFF 5 (GU) and 3 end of (AG). Some mutations
lengthen/shorten a protein by causing proteins to
change their length and function (i.e.
hemoglobin).
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Spliceosomes are RNA-protein complexes that cut
out unwanted pre-mRNA introns and link the
remaining exons into a single mRNA.
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Pre-mRNA is modified in many ways before exiting
the nucleus and finding a ribosome for translated
into a protein. Exons often become the
functionally distinct domains common to different
proteins with similar/dissimilar functions.
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RNAPI makes pre-rRNA. The 18S, 5.8S and 28S
rRNAs are cut from a single larger pre rRNA.
Methylations stabilize the rRNA
structure/sequences
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RNAPIII makes the tRNAs that carrying amino acids
to the nascent proteins chains that produced from
mRNA on a ribosome in the nucleus. tRNAs have a
clover-leaf shape and hair-pin curves made
possible by the RNA bases forming a DNA-like
double strand. Notice that pre-tRNA nucleotides
are removed, replaced and modified.