4. DNA Transcription and RNA processing Flashcards
Intergenic Regions:
Regulate Transcription
Each intergenic region contains a sequence that:
* Signals the end of transcription of the upstream gene
* Signals the start of transcription of the downstream gene
Transcription purpose:
mRNA molecule is transcribed from DNA template strand.
Where does Transcription occur?
Nucleus
Where does mRNA transcript go after being transcribed?
It is transported to the cytoplasm where it will used to generate a protein through translation.
Polypeptide Chain
resulting chain of translated amino acids
Differences between Transcription and DNA Replication
- Transcription bubble is much smaller (10-14bp)
- Initiation of RNA synthesis does not require a primer.
- No Ligase.
- Only helicase needed
- MAJOR DIFFERENCE: DNA unwinds at front of transcription bubble and then rewinds (glues back).
What synthesizes RNA in transcription?
RNA Polymerase. 5’-3’ direction.
* Catalyzes phosphodiester bonds
DNA Polymerase
- Synthesize DNA using DNA template.
- A, T, G, C
- Polymerizes 5’-3’
- Requires primer to start polymerization
RNA Polymerase
- Synthesizes RNA using DNA template
- A, U, G, C
- Polymerizes 5’-3’
- Reads template strand 3’-5’
- Does not require primer to start polymerization
SLIDE 14 DIAGRAM
Which RNA polymerase is involved in gene transcription?
RNA Polymerase II
Promoter Sequence:
Determines where template strand starts.
* RNA Pol II recognizes promoter at start of transcription.
Promoter has two main elements:
- Consensus Sequences: -35 and -10 (pribnow box)
- +1 Transcription start site
Consensus Sequences:
Attracts RNA Pol II
* -35 to -10 direction: determines orientation of RNA Pol II.
Sigma Factor:
Recognizes Pribnow Box and brings RNA Pol II complex to the promoter.
* DNA IS UNWOUND LOCALLY and RNA Pol II starts to create mRNA transcript.
What happens to sigma factor when transcription starts?
Sigma factor is released from holoenzyme and RNA Pol II continues chain elongation.
Holoenzyme:
Binds the promoter tightly and unwinds the double-stranded DNA, creating an open complex.
* Made up of several polypeptides
How does RNA Pol II start transcription?
RNA Transcription is initiated when core RNA Polymerase binds to the promoter with the help of sigma factor.
SLIDE 22 DIAGRAM
Terminator Sequence:
Tells RNA Pol II where to stop.
Three forms of RNA Polymerase:
- I: Produces rRNA
- II: Transcribes all protein-coding genes (makes mRNA)
- III: Transcribes all tRNA genes and one component of rRNA
Transcription in Eukaryotes: Differences with Prokaryotes:
1. The Presence of Nucleosomes. Chromatin must be relaxed.
- Nucleosome Remodeling Complexes (NRC)
-
Histone Modification Complexes (HMC)
Locally relax chromatin and make it accessible for transcription
Difference 2: Presence of multiple transcription factors
Examples:
2. 1. TFIIA and TFIIB
2. p53
3. c-Myc
4. CREB
5. c-FOS and c-Jun
RNA Pol II Recruitment:
- TFIIA/B - G rich sequence: On -35
- TBP (TATA Binding Protein): On -25
Difference 4: Presence of other regulatory sequences beyond promoter regions
- Enhancers or silencers: Can be found after the gene and even in an intron in the middle of the gene. Usually upstream the promoter.
Enhancer Sequences:
Promote transcription initiation. More common than Silencers.
Silencer sequences:
Inhibit Transcription
Activator Proteins:
Bind to enhancers and speed rate of transcription.
Inhibitors / Repressor Proteins:
Bind to silencers and slow transcription.
Coactivators:
“Adapter” molecules that integrate signals from activators and perhaps repressors.
Basal Transcription Factors:
Responds to injunctions from activators:
* Factors position RNA polymerase at start of transcription and initiate transcription process
Difference 5: Processing/maturing of RNA transcript
Pre-RNA molecule created by RNA Pol II processed in three steps:
1. 5’ Capping
2. Poly A tail formation
3. Splicing
5’ Capping:
PROTECTS starting 5’ from exonuclease activity.
* 7’ methylguanosine (methyl group on 7’ C) added to 5’ end of pre-mRNA
* 5’-5’ tri-phosphodiester bond
* Pre-mRNA is 5’ capped even before transcription is finished.
* Exonuclease cannot bind to active site to chew up the RNA strand
Poly A Tail Formation:
PROTECTS from 3’ exonuclease activity.
1. Series of Cleavage Factors recognize specific sequences around 3’ end.
2. Factors recruit PolyA Polymerase
3. PolyA Polymerase: CLEAVES later portion of 3’end. Starts adding multiple A to create a Poly A Tail.
4. PAB (polyA binding protein): recognize and bind to created polyA tail, further stabilizing 3’ end.
What is a good place to put PolyA Signal?
Exon.
* If put on intron, gene strand will be cut.
Splicing:
Removes Introns and keeps Exons.
Spliceosome:
Group of proteins that perform splicing.
Intron sequences:
- Start: GU sequence
- Finish: AG sequence
- Branch point
Intermediate Lariat (loop)
Initial loop that forms after intron branch point and initial GU sequence is recognized by splicosome machinery.
Formation of a Larait (detailed):
1. Intron is first cut at 5’ splice site (beginning of intron).
2. Lariat structure forms: 5’ end of intron is covalently linked to BRANCH POINT A residue through 2’ Oxygen of branch-point to 5’ end of intron. Phosphodiester bond forms between the 2’-5’.
Resolution of a Lariat:
- Spliceosome recognizes the AG terminal sequence.
- 3’ end of intron is then cut and 5’ and 3’ ends of exons are joined together through transesterification reaction.
SLIDE 51 DIAGRAM
Transesterification Reaction
OH group at 3’ end of exon ATTACKS phosphodiester bond at 3’ splice site. Results in covalent bonding of exons and release of lariat strucuture.
SLIDE 51 DIAGRAM
What is the evolutionary advantage to having exons and introns?
It allows alternative splicing
* One gene can give rise to multiple types of proteins.
* Form new forms of proteins without having to increase the number of genes
Alternative Splicing
The ability to create different types of mRNA transcripts from a single gene by changing how the pre-mRNA is spliced.
* Different combination of exons
