BCHM-3050 Lecture Notes - Lecture 6: Acetyl Group, Alternative Splicing, Repressor
27 May 2018
School
Department
Course
Professor
Review (replication)
A free strand of DNA is written 5' phosphate group to 3' OH group
Read left to right
○
•
Top strand: coding or + sense
Written 5' to 3'
○
•
Bottom strand: noncoding or antisense
3' to 5'
○
•
Sense v Antisense (transcription)
Antisense: template
Bottom strand - 3' to 5'
○
•
mRNA and DNA + sense strand are identical
Only difference = T is replaced by U and ribose is used
○
•
Operon Model
Predict that mRNA in bacteria would be:
A collection of metabolically active RNAs
○
Present in low abundance
○
Heterogenous in size
○
Complement sequence regions in DNA genome
○
•
Upstream: away from transcribed genes
Negative
○
•
Downstream: transcribed genes
Positive
○
•
Structural basics of mRNA
Promoter sequence: directs association of RNA polymerase
Begins transcription
○
•
Operator sequence: allow repressor protein to bind
Stop or allow transcription
○
Inducer binds to repressor causing transcription to occur
○
•
Sequence of operator in reference to promoter may:
Overlap with promoter
○
Be within the promoter sequence
○
Lie upstream from promoter
○
•
Consensus sequence: nucleotides most often found at each position
•
DNA foot printing: good for finding unique promoter sequences for
regulatory proteins
•
TATA box in eukaryotes
-10 region in prokaryotes
○
•
TTGACA in eukaryotes
-35 region in prokaryotes
○
•
Detection of mRNA w/ pulse label and sediment
mRNA has short half life in bacteria
2-3 minutes
○
Would be a waste of resources to have them for a prolong period of
time
○
•
Signal of RNAs disappear overtime
•
Three distinct RNA polymerase in Euk v one in Prok
Bacteria: one RNA polymerase
5 subunits
Alpha: interacts with regulatory protein and promoter elements
§
Beta: elongation
§
Beta prime: DNA binding
§
Sigma: promoter recognition***
§
Omega: promotion of enzyme assembly
§
○
•
Eukaryotes: three RNA polymerase
Allow for specificity
○
•
RNA polymerase Holoenzyme structure
Molecular weight: ~450,000 Da
•
Sigma factor: plays role in directing RNA polymerase to the promoter on
the gene of interest
w/o = polymerase loses specificity
○
•
Overview of Transcription
Initiation 1.
Elongation 2.
Termination 3.
Initiation: binding of RNA polymerase to promoter sequence
Step #1: form closed complex region
Template can not be unwound
§
○
Step #2: open or unwound complex binds to -10 region
○
Create "transcription bubble"
○
•
Elongation: addition of rNTPs in 5' to 3' direction
Like polymerase in replication
○
•
Termination: recognition of transcription terminator sequence
Release RNA polymerase and mRNA strand
○
•
Initiation to Elongation
Elongation begins by binding the first rNTP
•
DNA bends into open promoter complex
•
DNA continuously unwound and rewound in bubble
Sigma factor dissociates
○
•
Backtracking and Discontinuity
Seen in replication
Correct mistakes
○
•
3' RNA nucleotide can be repositioned into the active site
•
Pausing and backtracking controls transcription rate and fidelity
•
Factor independent termination of transcription
Termination: factor independent and factor dependent
•
Factor independent: 2 GC rich segments form a stem loop
GC rich segments: slow transcription and allows loop to form
○
Weaken protein- DNA complex
○
'A' rich segment: further weakens interaction
Cause release and termination
§
○
•
Factor dependent: require rho protein
Homohexameric
○
•
Transcription polymerases
RNA polymerase I: major ribosomal RNA genes
•
Pol II: protein coding genes and small RNA genes
•
Pol III: small RNA genes
•
Transcription factors: TFI, TFII, TFIII
Match polymerases
○
Wont accompany Pol during elongation
○
•
TFIIA: zinc finger protein
Helps with binding
•
One of three transcription factors required by pol III
•
Alpha helix of zing finger protein fits major groove of DNA
•
Can be strung together
•
DNA loop = bring activator close
Pol II interacts with many factors:
TATA binding proteins: help DNA bend 90 degrees
○
TFIIA, -B, -E, -F, and -H
○
•
TFIIB: has beta linker element
Opens DNA
○
•
Mediator: communicate between enhancer region and proteins bound at
promoter
•
High level of histone acetyl associated with high Transcription
Acetylation: weaken interaction b/w histone and Dna in chromatin
Lessen the hold
○
Neutralization
○
•
Termination in Eukaryotes
Pol II: transcribe post end of gene
Pass one or more TTATT signals
○
•
Pre-mRNA with AAUAA signal is cleared 11 to 30 units downstream of
site
•
3' Poly A tail: added by poly A polymerase
Stabilize mRNA
mRNA is usually degraded starting at the 3' end
§
○
Long tail = long half life
○
•
5' cap: 7-methylguanasine cap
Extend life and guide ribosome to 5' end
○
•
Splicing
snRNPs aid in pre-mRNA loop formation
•
2' OH of branch pt.: nucleophilic attack on phosphate of the base at the 5'
splice site
Form lariat and release 5' exon
○
•
3' OH of 5' exon attacks phosphate of first nucleotide of 3' exon forming
splice mRNA
•
snRNP + pre-mRNA = spliceosome
•
Alternative splicing: allow one gene to specify multiple proteins
Ex. Tropomyosin
○
•
Chapter 24: Transcription
Sunday, May 27, 2018
3:04 PM
Review (replication)
A free strand of DNA is written 5' phosphate group to 3' OH group
Read left to right
○
•
Top strand: coding or + sense
Written 5' to 3'
○
•
Bottom strand: noncoding or antisense
3' to 5'
○
•
Sense v Antisense (transcription)
Antisense: template
Bottom strand - 3' to 5'
○
•
mRNA and DNA + sense strand are identical
Only difference = T is replaced by U and ribose is used
○
•
Operon Model
Predict that mRNA in bacteria would be:
A collection of metabolically active RNAs
○
Present in low abundance
○
Heterogenous in size
○
Complement sequence regions in DNA genome
○
•
Upstream: away from transcribed genes
Negative
○
•
Downstream: transcribed genes
Positive
○
•
Structural basics of mRNA
Promoter sequence: directs association of RNA polymerase
Begins transcription
○
•
Operator sequence: allow repressor protein to bind
Stop or allow transcription
○
Inducer binds to repressor causing transcription to occur
○
•
Sequence of operator in reference to promoter may:
Overlap with promoter
○
Be within the promoter sequence
○
Lie upstream from promoter
○
•
Consensus sequence: nucleotides most often found at each position
•
DNA foot printing: good for finding unique promoter sequences for
regulatory proteins
•
TATA box in eukaryotes
-10 region in prokaryotes
○
•
TTGACA in eukaryotes
-35 region in prokaryotes
○
•
Detection of mRNA w/ pulse label and sediment
mRNA has short half life in bacteria
2-3 minutes
○
Would be a waste of resources to have them for a prolong period of
time
○
•
Signal of RNAs disappear overtime
•
Three distinct RNA polymerase in Euk v one in Prok
Bacteria: one RNA polymerase
5 subunits
Alpha: interacts with regulatory protein and promoter elements
§
Beta: elongation
§
Beta prime: DNA binding
§
Sigma: promoter recognition***
§
Omega: promotion of enzyme assembly
§
○
•
Eukaryotes: three RNA polymerase
Allow for specificity
○
•
RNA polymerase Holoenzyme structure
Molecular weight: ~450,000 Da
•
Sigma factor: plays role in directing RNA polymerase to the promoter on
the gene of interest
w/o = polymerase loses specificity
○
•
Overview of Transcription
Initiation
1.
Elongation
2.
Termination
3.
Initiation: binding of RNA polymerase to promoter sequence
Step #1: form closed complex region
Template can not be unwound
§
○
Step #2: open or unwound complex binds to -10 region
○
Create "transcription bubble"
○
•
Elongation: addition of rNTPs in 5' to 3' direction
Like polymerase in replication
○
•
Termination: recognition of transcription terminator sequence
Release RNA polymerase and mRNA strand
○
•
Initiation to Elongation
Elongation begins by binding the first rNTP
•
DNA bends into open promoter complex
•
DNA continuously unwound and rewound in bubble
Sigma factor dissociates
○
•
Backtracking and Discontinuity
Seen in replication
Correct mistakes
○
•
3' RNA nucleotide can be repositioned into the active site
•
Pausing and backtracking controls transcription rate and fidelity
•
Factor independent termination of transcription
Termination: factor independent and factor dependent
•
Factor independent: 2 GC rich segments form a stem loop
GC rich segments: slow transcription and allows loop to form
○
Weaken protein- DNA complex
○
'A' rich segment: further weakens interaction
Cause release and termination
§
○
•
Factor dependent: require rho protein
Homohexameric
○
•
Transcription polymerases
RNA polymerase I: major ribosomal RNA genes
•
Pol II: protein coding genes and small RNA genes
•
Pol III: small RNA genes
•
Transcription factors: TFI, TFII, TFIII
Match polymerases
○
Wont accompany Pol during elongation
○
•
TFIIA: zinc finger protein
Helps with binding
•
One of three transcription factors required by pol III
•
Alpha helix of zing finger protein fits major groove of DNA
•
Can be strung together
•
DNA loop = bring activator close
Pol II interacts with many factors:
TATA binding proteins: help DNA bend 90 degrees
○
TFIIA, -B, -E, -F, and -H
○
•
TFIIB: has beta linker element
Opens DNA
○
•
Mediator: communicate between enhancer region and proteins bound at
promoter
•
High level of histone acetyl associated with high Transcription
Acetylation: weaken interaction b/w histone and Dna in chromatin
Lessen the hold
○
Neutralization
○
•
Termination in Eukaryotes
Pol II: transcribe post end of gene
Pass one or more TTATT signals
○
•
Pre-mRNA with AAUAA signal is cleared 11 to 30 units downstream of
site
•
3' Poly A tail: added by poly A polymerase
Stabilize mRNA
mRNA is usually degraded starting at the 3' end
§
○
Long tail = long half life
○
•
5' cap: 7-methylguanasine cap
Extend life and guide ribosome to 5' end
○
•
Splicing
snRNPs aid in pre-mRNA loop formation
•
2' OH of branch pt.: nucleophilic attack on phosphate of the base at the 5'
splice site
Form lariat and release 5' exon
○
•
3' OH of 5' exon attacks phosphate of first nucleotide of 3' exon forming
splice mRNA
•
snRNP + pre-mRNA = spliceosome
•
Alternative splicing: allow one gene to specify multiple proteins
Ex. Tropomyosin
○
•
Chapter 24: Transcription
Sunday, May 27, 2018 3:04 PM
Document Summary
A free strand of dna is written 5" phosphate group to 3" oh group. Bottom strand - 3" to 5" mrna and dna + sense strand are identical. Only difference = t is replaced by u and ribose is used. Inducer binds to repressor causing transcription to occur. Sequence of operator in reference to promoter may: Consensus sequence: nucleotides most often found at each position. Dna foot printing: good for finding unique promoter sequences for regulatory proteins. Detection of mrna w/ pulse label and sediment mrna has short half life in bacteria. Would be a waste of resources to have them for a prolong period of time. Three distinct rna polymerase in euk v one in prok. Alpha: interacts with regulatory protein and promoter elements. Sigma factor: plays role in directing rna polymerase to the promoter on the gene of interest w/o = polymerase loses specificity. Initiation: binding of rna polymerase to promoter sequence.
