CHANGE: First trimester exam will be next Tuesday, Sept 30.
This change is to reduce conflict some students have with the organic chemistry exam schedule, and will also allow students to attend their recitation sessions prior to the exam. Other exams in Bot132 will also be moved to Tuesdays, and will cover the same topical information as originally scheduled.

Some information about the first trimester exam can be seen on the see Exams page

Transcription of Genes, Part II

Transcription in eukaryotes

Recall, four main types of RNAs:

• mRNA (protein coding)
• tRNA (many types, all similar in structure) (secondary structure of RNA)
• rRNA (several distinct types, highly conserved in evolution) (secondary structure of RNA)
  • Sucrose gradient centrifugation to isolate rRNA based on their size
  • Prokaryotes: 23S, 16S, 5S
  • Eukaryotes: 28S, 18S, 5.8S, 5S
• snRNA (diverse roles, such as in splicing)

In euk's, three type of RNA polymerase are important

• RNA Pol I
  • 18S, 28S, 5.8S rRNAs, in nucleolus
• RNA Pol II, in nucleoplasm
  • mRNAs, some snRNAs

• RNA Pol III, in nucleoplasm
  • tRNAs, 5S rRNA, other snRNAs

  RNA polymerase I, II, III are composed of many subunits each (recall E. coli RNA Pol I has just 4 subunits). Further, eukaryotic RNA pol's differ in WHERE they are found, the genes they transcribe, and sensitivity to toxin.

The fungal toxin adamant is highly toxic to RNA-Pol II transcription

• RNA Pol I: 1 mg/ml inhibits
• RNA Pol II: 0.01-0.05 ug/ml inhibits
• RNA Pol III: 10-25 mg/ml inhibits
• See chart

mRNA synthesis by RNA pol II

Promotors of eukaryotic mRNA genes:

Basal promotor elements

• TATA box at approx position -25
• Initiator (Inr) element near transcriptional start site (TSS)

Promotor proximal elements between -50 and -200

• CAAT box around -75 relative to TSS
• GC box (GGGCGG) approx -90 relative to TSS

Most promotors are complex, with many overlapping and redundant cis elements, some acting to promote, others acting to repress transcription

Basal transcription factors

• General factors used on many different gene promoters
• Nomenclature: TFIID, TFIIA, etc

Enhancers

• Sequences that can act at a distance (up to several kbp away) to promote transcription of linked genes
• Can act upstream or downstream of an affected gene
• Orientation independent

Silencer elements

Initiation of transcription

• A number of proteins participate
• Proteins are specific to a type of RNA polymerase
• Nomenclature: numbers by PNA pol type, lettered by discovery (e.g. TFIID: 4th TF discovered for PolII transcription
• Binding of TFs occurs in a regular sequence, prior to recruitment of RNA pol core complex

In eukaryotic Pol II transcribed genes, no termination sequence is present

• RNA Pol II continues past coding sequence, sometimes making a primary transcript thousands of nucleotides longer than the mature RNA will be
• Sequences within the primary transcript are recognized by 3' end-processing enzymes
  • polyA consensus sequence - a sequence within the primary transcript
  • CPSF -cleavage and polyA specificity factor
  • PAP -polyA polymerase (uses ATP)
  • Other protein factors involved too
• These enzymes cleave the primary transcript at a defined site, then enzymatically add on dozens to hundreds of A nucleotides to form the "poly-A tail"

Transcript composed of 5' UTR, CS, 3' UTR

Primary RNA product is a precursor, and processing is required to produce mature mRNA

5' and 3' modification of mRNAs

• Capping of 5' end of mRNA
  • Addition of 7-me-G, methylation of adjacent ribose sugars
  • Role in ribosome binding
• Polyadenylation of 3' end of transcript
  • several hundred A's added enzymatically to transcript
  • Role in mRNA stability, termination of transcription

Intron excision

• Evidence through comparison of cDNA and genic sequences
• Sequences within precursor RNA trigger splicing
• Typical junction is marked by 5' GU, then AG-3' and local context
• Splicing occurs in nucleus mediated by protein:snRNA complex "spliceosome"

RNA editing may alter sequence of mRNA in unusual cases

Key differences between in transcription in promotes and eukaryotes

Prok: transcription/translation coupled and in same place
Euk: transcription nuclear; translation cytoplasmic

Prok: One RNA polymerase
Euk: Several RNA pol's

Prok: RNA product used immediately for translation
Euk: Precursor matured through splicing, 5' capping, 3' polyA addition

Prok: Messages may be polycistronic
Euk: Messages typically monocistronic

Notes:

We will not cover here these topics from Chapter 5: spliceosome structure/rxns, autocatalytic splicing, additional details about RNA PolI, RNA Pol III transcription (other than those already mentioned)

We will cover ribosome structure/composition/function next Tuesday.

Next: Ribosome structure/translation

Please work through the following problems from Chapter 5 and lecture.

Problems: 5.5, 5.6, 5.10, 5.19, and:

Contrast sucrose gradient centrifugation from CsCl gradient centrifugation. What is the basis for separation of molecules using these two types of purification techniques?

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