Lecture 20: Transcription & RNA Metabolism Flashcards
Intro Facts
Where does Transcription and Translation occur in prokaryotic cells?
Eukaryotic cells?
Prokaryotic - everything occurs in the cytoplasm
Eukaryotic
- Transcription & Processing/Transportation
= Nucleus
- Translation = in the Cytoplasm
Intro Facts
What is transcription?
What is the purpose of transcription?
- Transcription is the process of copying a segment of DNA into RNA
- The purpose of transcription is to make RNA copies of genes
Intro Facts
How many DNA serve as a template in transcription?
Only one DNA will serve as a template
Intro facts
What is the template strand?
What is the non template/coding strand?
Template: The strand that is complimentary to RNA transcript
Non template: the same sequence as RNA (with T instead of U)
Transcription vs. DNA replication
How much of the genome is copied in Transcription vs. DNA replication?
Transcription: One/particular group of genes
DNA replication: whole genome
Transcription vs. DNA replication
What is the template used in Transcription vs. DNA replication?
Transcription: only one strand serves as the template
DNA replication: both strands will serve as template
Transcription vs. DNA replication
Which processes use primers?
Transcription does not use primers
DNA replication uses primers
Transcription Basics
Are polymers built continuously?
Yes, polymers are built continuously (processive)
Transcription Basics
In what direction is RNA synthesized?
Describe the relationship between the template and RNA strand
5’ -> 3’
- They are anti-parallell
Transcription Basics
What is RNA Polymerase?
Enzymes that catalyzes the synthesis of RNA by reading the template strand
Transcription Basics
Does RNA polymerase require a primer?
What does RNA polymerase use?
- Does not require a primer
- Uses ribonucleotides
Essential Components of a Gene
What are the 4 essential components of a gene?
1) Promoter
2) Transcription factors
3) RNA Polymerase
4) Terminator
Essential Components of a Gene
What is the promotor region?
Sequence of DNA where transcription proteins assemble
Essential Components of a Gene
What specific machinery do we need at the promoter?
- Transcription factors
- RNA Polymerase
Essential Components of a Gene
What do transcription factors do?
They are proteins that bind to promoters to direct RNA synthesis
MAIN PURPOSE: Increase affinity of RNA polymerase at the promoter region
Essential Components of a Gene
What is the terminator region?
Sequence of DNA where transcription ends
Transcription Phases
What are the 4 phases of transcription?
What is its nemonic?
1) Assemble
2) Initiation
3) Elongation
4) Termination
Aliens Invade Earth Tonight
Transcription Phases
Which phases of transcription are very different in prokaryotes vs. eukaryotes?
Initiation & termination
Transcription Phases
What are transcription factors typically referred to as in prokaryotes?
Sigma factors
Transcription Phases
What are sigma factors?
Proteins that bind to specific DNA sequences in the promoter and increase affinity for RNA polymerase
Transcription Phases
What are consensus sequences?
DNA sequences that transcription factors and polymerases repeatedly assemble at
Transcription Phases
Where do transcription factors, sigma factors, and polymerases assemble?
They assemble at the promoter
Transcription Phases
What are the steps of initiation?
1) Initiation occurs at the promotor sequence
2) Transcription factors, sigma 70 & RNAPs assemble at the promoter region and forms a closed complex
3) Transcription bubble forms and RNAP forms open complex
4) Transcription is initiated
Transcription Phases
What are the main things that occur during elongation?
What are the steps of elongation?
Main Occurrences
- Clearing the promoter
- RNA polymerase builds the RNA strand by complementing the template strand
Steps
1) Sigma 70 disassociates
2) Sigma 70 is replaced by NusA
3) Transcription terminates and NusA disassociates
Transcription Phases
Super coils form during the elongation process
In what direction do they form?
What kind form?
Positive supercoils in the direction of transcription
Negative supercoils in the opposite direction of transcription
Transcription Phases
What is the cost of putting in a new nucleotide during elongation?
2 ATP equivalents
Transcription Phases
What is the main thing that occurs during termination?
RNA polymerase dissociates from DNA strand, releasing newly synthesized RNA polymer
Transcription Phases
What are the two different termination methods that occur in prokaryotes
- ρ-independent
- ρ-dependent
Transcription Phases
Describe the difference between ρ-independent and ρ-dependent
ρ-independent
- relies on a repeating palindromic sequences at the termination sequence
- RNA transcribed from these sequences fold onto itself
ρ-dependent
- relies on ρ helicase, a hexamer protein that binds to the rut element and travels fowards, disassociating the elongation machinery
Eukaryotic vs. Prokaryotic Transcription
What are the 3 key differences between the two transcription methods?
1) There are many MORE transcription factors
called general transcription factors in
assembly, not one signal sigma factor
- However their job is still to increase affinity of promoter to RNA polymerase
2) C-terminal Domain (CTD) on RNA polymerase
is phosphorylated to start elongation
3) In the same vein, elongation is terminated by
dephosphorylation of RNA polymerase by
termination factors 12
Eukaryotic vs. Prokaryotic Transcription
What are the RNAPs present in Bacteria/Archea?
only one form of RNAP
Eukaryotic vs. Prokaryotic Transcription
What are the RNAPs present in Eukaryotes?
List them
Multiple different types
- RNAP I
- RNAP II
- RNAP III
- RNAP IV/RNAP V
Eukaryotic vs. Prokaryotic Transcription
What are the each specific RNA polymerase responsible for?
For producing different types of RNAs
Eukaryotic vs. Prokaryotic Transcription
What RNA(s) does RNAP I produce?
rRNA
Eukaryotic vs. Prokaryotic Transcription
What RNA(s) does RNAP II produce?
mRNA, snRNA, miRNA
Eukaryotic vs. Prokaryotic Transcription
What RNA(s) does RNAP II produce?
mRNA, snRNA, miRNA
Eukaryotic vs. Prokaryotic Transcription
What RNA(s) does RNAP III produce?
tRNA
Eukaryotic vs. Prokaryotic Transcription
What RNA(s) does RNAP IV/V produce?
siRNA (plants)
Eukaryotic vs. Prokaryotic Transcription
How many genes are transcribed in Prokaryotes?
polycistronic
- Transcribe multiple genes at once
- Genes are transcribed as operons
Eukaryotic vs. Prokaryotic Transcription
How many genes are transcribed in Eukaryotes?
Monocistronic (ONE gene at a time)
Eukaryotic vs. Prokaryotic Transcription
What is unique about RNA polymerase II?
Phosphorylation state of the C-terminal domain (CTD) changes with each stage of transcription
Post Translational Processing
What are the 3 Steps to Process RNA?
When do these steps occur?
1) 5’ end is capped
2) Exon sequences are spliced together
3) Poly-adenylated tail (Poly-A) is added to the 3’ end
These steps occur alongside transcription, they are co-transcriptional
Post Translational Processing
Why are Eukaryotic mRNAs capped?
1) To protect from ribonuclease
2) Enable binding to cap-binding proteins and ribosomes
Post Translational Processing
What occurs during the capping of the 5’ end?
What are the results?
- Add 7-methylguanosine to 5’ end
- Enhanced stability
- Roles in processing/translation
Post Translational Processing
What do Poly (A) tails contribute to?
Roles in stability and translation
Post Translational Processing
What are primary transcripts spliced into?
a continuous coding sequence
Post Translational Processing
What are introns?
Sequences of RNA in between expressed regions of the transcript
Post Translational Processing
What are exons?
Sequences of RNA transcript that code for protein
Post Translational Processing
How do introns and exons vary in length?
Introns vary widely between 50-700,00 nucleotides
~1800 nucleotides on average
Exons are relatively short less than 1000 nucleotides
~100-200 on average
Post Translational Processing
What are the 2 splicing mechanisms?
1) Self-Catalyzed splicing
2) Spliceosome Mediated Splicing
Post Translational Processing
Describe self catalyzed splicing
- Where does it occur?
- What are the 4 steps?
Occurs in RNA but NOT DNA
1) Driven by the higher reactivity of RNA given by the 2’ OH
2) 2’ OH specific Adenosine performs a nucleophile attack of the 5’ splice site, forming lariat
3) Exposed 3’ OH of 5’ exon (the base at the 5’ splice site was “stolen”) now attacks the other side of the lariat
4) The lariat disassociated and the two ends of the exon are ligated together
Post Translational Processing
What do Eukaryotes utilize on order to splice transcripts?
Spliceosome
Post Translational Processing
What is a Spliceosome?
A large complex made up of small nuclear ribonucleo Proteins (snRNPs)
Post Translational Processing
What do snRNPs contain?
specific small nuclear RNA to interact with specific sequences within pre-mRNA
Post Translational Processing
What do snRNPs do?
Binds to intron and assemble to entire spliceosome to cut that region out
Post Translational Processing
What do poly-A binding proteins regulate?
They regulate RNA export, breakdown, and aid in translation to protein
Post Translational Processing
What 2 steps occur during the poly a tail addition?
1) Primary transcript is trimmed, leaving a consensus sequence at the 3’ end
2) Polyadenylate polymerase adds 30 (yeast) to 100 (animals) A residues
Post Translational Processing
What is alternate splicing?
What does it result in?
the same “raw” transcript can result in two different mature transcripts
these two different transcripts translate into two different protein sequences with different downstream effects
Post Translational Processing
What does alternative splicing allow for?
Usually allows for tissue specific responses of a gene
Allows more possible “outputs”
Post Translational Processing
How is alternative splicing used to treat Spinal Muscular Atrophy?
- SMN1 protein is produced to mainatin motor neurons
- SMA patients have a mutation in SMN1 gene - no protein produced leads to degeneration of neurons
- Nusinersen effectively hides SMN2 RNA, affecting alternative splicing, producing a functional protein
Different forms of RNA
Why is rRNA processed?
Ribosomal RNA is processed to produce proper ribosome structure
Different forms of RNA
How is rRNA formed?
1) Modify many RNA nucleotides
2) Cut it into smaller pieces
1) Pieces come together to form 3 dimensional structure
Different forms of RNA
Why is tRNA processed?
tRNA is processed to produce specific structures
Different forms of RNA
How is tRNA formed?
A single strand of RNA forms intramolecular base pairs to fold into specific shapes
Different forms of RNA
What helps govern the level of gene expression?
the cellular concentration of mRNA
Different forms of RNA
Describe the relationship between the rate of mRNA synthesis and the rate of mRNA degredation
When synthesis = degradation, mRNA remains constant (steady state)
Different forms of RNA
What
Different forms of RNA
What does miRNA do?
What is it utilized for?
Fine tunes gene expression
Utilized to target specific sequences for either silencing or actual degradation
Different forms of RNA
What is miRNA made of?
Who utilizes miRNA?
22 nucleotide RNA oligomers
Plants and Animals
Different forms of RNA
What is unique about the structure of miRNA?
Near perfect complement mRNA sequences and can inhibit translation or degrade mRNA
RNA Degradation
How is mRNA degraded in prokaryotes?
It is degraded by ribonuclease.
Essentially: Individual proteins responsible for cleaving the RNA backbone and chewing up the polymer
- They utilize enzymes known as endonuclease
- They cut in the middle portion of the mRNA
- Exonuclease’s can come from the end and completely digest these particles themselves
RNA Degradation
How is mRNA degraded in Eukaryotes?
What enzyme do they utilize?
They utilize a very specific enzyme - endonuclease
RNA Degradation
What is endonuclease?
What does it do?
It’s a large protein complex
It unwinds and feeds mRNA through a nuclease for cleavage
RNA and the origins of life
What is a Ribozyme?
What does it suggest?
RNAs that fold into highly specific 3D shapes and catalyze reactions
Suggests that perhaps there was an old version of enzymes made of RNA
RNA and the origins of life
What is in the catalytic core of ribosomes composed of?
The catalytic core is composed of RNA
