RNA pol II and mRNA

1
Lecture 28

• RNA pol II and mRNA
• Regulation by 2 control elemens
• Promoter
• TATA box bound by TFIID -composed of TBP and TAFs
  (TATA-binding associated factors)
• Enhancers
• DNA sequences that can bind multiple regulatory
  proteins
• Can be far away orientation doesnt matter
• Ways of studying enhancer function

2
Lecture 28

• Motifs found in DNA-binding proteins
• Zinc fingers
• Protein structural domain that binds a Zn2 ion
  though interaction with 2 alpha helices and two
  beta sheets
• Leucine zippers
• Protein structure motif in which leucine residues
  lie n one face of an alpha helix, allowing it to
  interact with a similar protein forming either a
  homo- or hetero-dimer
• HLH motif-
• Alpha helices that er spaced so as to fit into
  the DNA grooves

3
Lecture 28

• Modifications of mRNAs
• 5 capping
• Only added onto pol II transcripts
• GTP added in reverse orientation, methyl groups
  are also added
• Serves to position the ribosome and to stabilize
  the transcript
• Poly A tailing
• Polyadenylation site (AAUAAA) in sequence signals
  the end of transcription, nuclease cuts
  downstream from that
• Poly A polymerase adds on A residues
• Useful for isolating mRNAs

4
Lecture 29

• Splicing
• Types I and II-self splicing, rRNA and a few
  unusual mRNAs
• Type III- generally eukaryotic mRNAs
• Type III splicing involves snRNPs made up of
  small RNAs and proteins-gt spliceosome
• 3 important snRNPs
• U1 binds 5 splice site
• U2 binds branch point
• U3 binds 3 splice site

5
Lecture 29

• Splicing involves sequences at the splice sites
  and at the branch point
• Mechanism of splicing- steps involved such as
  lariat formation and 5-2 linkage
• self splicing- requirements
• Alternative splicing
• How does it occur
• How does it affect coding sequences?

6
Lecture 30

• More examples of the regulation of splicing
• Repressor and activators determine which splice
  sites are used
• Example- calcitonin/neuropeptide gene
• How do enhancers aid in splicing at weak sites?
• Alternative promoters are also used sometimes-
  result in alternative 5 first exons

7
Lecture 30

• Example of regulation of gene expression- stripes
  in Drosophila
• technique used to determine enhancer elements-
  clone DNA fragments upstream of reporter genes
  and assay their expression in flies
• This allows you to determine the impact that
  activators and repressors have and is affected by
  their spatial localization
• thus,, in some tissues the activators are present
  and you get gene expression, in other tissues the
  repressors are present and no gene expression is
  detected

8
Lecture 30

• Another way to analyze gene expression
• Microarrays
• Involves preparing mRNA from two populations of
  cells that you want to compare (usually normal
  and mutant/diseased/abnormal)
• mRNA is made into labelled cDNA pools
• Hybridized to microarrays that contain DNA from
  all known genes
• The level of hybridization tells you whether the
  gene is expressed at high levels or low levels
  (since cells that make more mRNA from a
  particular gene will have more labelled probe for
  that gene, thus more signal)- can detect changes
  in gene expression between the two populations of
  cells

9
Lectures 31-32

• Translation
• mRNA contains sequences that encode protein (open
  reading frame)
• Relationship of DNA strands to mRNA and coding
  sequences
• Involves 3 RNAs
• mRNA
• tRNA
• rRNA

10
Lectures 31-32

• tRNA structure and function
• tRNA synthetases charge the tRNA with its
  corresponding amino acid to generate amino-acyl
  tRNA
• Anticodon and wobble hypothesis
• Genetic code
• 20 amino acids
• 3 nucleotides per codon
• How was the code cracked?
• Random copolymers, synthetic polymers of known
  sequence

11
Lectures 31-32

• Genetic code
• Degeneracy
• How do the sequences of the codons relate to the
  anticodon? Wobble site? What if a mutation occurs
  in one of the sites? What is the effect?
• Ribosome
• 2 subunits (contain many proteins and rRNA)
• Prokaryotes- 30s, 50s-gt70s
• Eukaryotes- 40s, 60s-gt80s

12
Lectures 31-32

• Translation
• Initiation
• Requires initiation factors, bring the mRNA
  together with the ribosome subunits and bring in
  the first fMet amino acid
• Prokaryotes- Shine Delgarno sequence 5-10 bp
  upstream from start, basepairs with 16s rRNA
• Eukaryotes- 5 cap is recognized by ribosome and
  then first AUG is used as start

13
Lectures 31-32

• Elongation
• Involves 3 sites in ribosome- A, P, E
• EF-Tu brings aminoacyl tRNA to A site
• Peptide bond formation catalyzed by rRNA in 50s
  subunit
• Termination
• Requires release factors that bind to stop codons
  and release peptide chain

14
FINAL EXAM

• Wednesday April 17th
• 830 a.m.
• B 9201
• Bring your student ID with you!
• No ID, no mark!
• Extreme illness- contact me (or have someone else
  do it) the day of the exam- no later than noon.