Lecture 14: Transcription Flashcards
Central Dogma
the flow of information is from DNA to RNA to protein
DNA transcripted to
RNA
RNA translated to
protein
Transcription
the process of generating an RNA copy of a DNA molecule
Similarities to DNA Replication
- Requires a single strand DNA template
- Enzymes make nucleic acid chains with phosphodiester bonds
- Chains are extended from 5 prime to 3 prime direction (using 3’-OH group)
- New strand made is complementary to a template strand
Transcription uses
Ribonucleotides
Transcription uses what sugar
Uracil
Transcription: a _______ portion of the genome is utilized
small
Transcription occurs at what times?
Various times as the gene product is needed
RNA product is
displaced from template
RNA polymerase does not need a
primer
______ strand of DNA is used as a template during transcription
One
One to _________ are made during transcription
several thousand copies
tRNA
transfers amino acids to ribosomes for protein synthesis
mRNA
Contains the coding sequences for cellular proteins. mRNA is the only species of RNA that is translated into protein.
rRNA
A component of ribosomes (bound by ribosomal proteins to form the mature ribosome).
Provides structural support and catalyzes the chemical reaction in which amino acids are covalently linked to one another (peptidyl transferase activity)
Eukaryotes only
snRNAs (small nuclear RNAs): involved in mRNA processing (splicing)
For some genes, their functional products are RNA rather than__________. These genes are referred to as.
protein. Noncoding RNA genes (ncRNAs). They represent about half of all identified human genes.
What are two groups of noncoding RNAss?
- Housekeeping RNAs
- Regulatory RNAs
House keeping RNAs
- rRNA
- tRNA
- snRNA
- snoRNA
snRNAs
(small nuclear) involved in making mature mRNAs
snoRNAs
(small nucleolar), involved in modifying rRNAs
Regulatory RNAs
- MicroRNAs
- Long ncRNAs
MicroRNAs (miRNAs)
involved in regulation of protein production
Long ncRNAs (lncRNAs)
involved in gene regulation and gene slicing
siRNAs
small interfering RNAs
piRNAs
piwi-associated RNAs
Template strand
non-coding strand
Non template strand
coding strand
RNA is transcribed in what direction?
5’ to 3’ direction with respect to the new molecule being synthesized. Template DNA reads from 3’ to 5’ direction.
RNA has the same sequence: except for? As the?
except for the U’s as the non-template strand hence, the non-template strand is referred to as the “coding strand”
What is the transcription start site?
+1: transcription start
AUG: start of
What three letters codes for the start of translation?
AUG
UTR (untranslated region)
does not code for the functional proteins but is important for mRNA stability and translation regulation
Upstream
direction opposite of way RNApol will travel
Downstream
direction RNApol will travel
promoter
DNA sequence where RNApol and transcription factors bind and initiate transcription
The +1 is NOT the same as
the first codon of the protein coding region
Prokaryotic Transcription Events (3)
- Initiation
- Elongation
- Termination
Initiation
- Binding of RNApol
- Open-promoter complex
Elongation
- Conformation changes in RNApol (o subunit)
- Start of transcription
- Unwinding, elongation, and re-annealing
Termination
- Use of terminators
- Stop & release of newly synthesized transcript (RNA)
Prokaryotic transcription is performed by a what RNA polymerase?
Single
RNA polymerase how many subunits? How many catalytic and what else?
6 subunits (4 catalytic subunits and a single regulatory subunit known as a sigma (o) subunit/factor)
Prokaryotic RNA polymerase holoenzyme is made up of
- Catalytic subunits
- Regulatory subunits
Catalytic subunits (core polymerase)
-2 alpha subunit
-1 beta subunit
-1 beta prime subunit
-1 w subunit
Regulatory subunit
1 o (sigma) subunit
Catalytic subunits catalyzes RNA synthesis only
when dissociated from a sigma subunit
The regulatory subunit does what?
Facilitates polymerase binding to DNA at 35 & -10 promoter sequence/region
Promoter sequences
DNA sequences that define the start sites of RNA synthesis (where transcription of a gene by RNA polymerase begins)
Prokaryotic RNA polymerase initially binds to a what? Recognizes what? Where?
Binds to a promoter and recognizes 10 and 35 base pairs upstream of the actual start site of transcription (+1 site). These are called -10 and -35 elements.
The -10 and -35 elements are composed of how many nucleotides each?
Six
-10 contains ONLY
adenines and thymines
-35 contains are
AT rich
The nucleotide sequences at the -10 and -35 elements are fairly
conserved at various genes (consensus sequences), ensuring that even though gene sequences may differ, the polymerase will always recognize the appropriate binding site
Prokaryotic Transcription Overview (first two steps)
- Holoenzyme binds to the DNA. Sigma unit is responsible for recognizing the -10 and -35 sequences and positioning the holoenzyme to the promoter sites.
- Sigma (o) subunit unwinds DNA around the initiation site –> “open promoter complex.” RNA polymerase can separate dsDNA without recruiting an external helicase.
(Initiation)
Transcription Steps 3 and 4
- Sigma subunit is released from the polymerase
- With release of sigma subunit, the polymerase can start the elongation process
(Initiation)
Elongation
During elongation, the polymerase remains associated with its template.
As it travels, the polymerase unwinds the template DNA ahead of it and rewinds the DNA behind it, minimizing the unwound region of ~15 base pairs.
Unlike replication
The single stranded DNA state needs to be limited. Single-strand DNA binding proteins are not needed.
Termination
RNA synthesis continues until the polymerase encounters a termination signal (DNA sequence), at which point transcription stops.
Then RNA is released from the polymerase, and the enzyme dissociates from the DNA template.
Prokaryotic transcription can be terminated in two ways
- Rho-dependent
- Rho-independent
Rho-independent
-Rho protein is NOT needed
-Utilizes palindromic sequence on DNA that yields RNA transcript that can form a “hairpin”
mRNA sequence (not DNA sequences) causes to form
hairpin structure
Hairpin created with
RNA sequences AND stretches of uracil-adenine pairing disrupts RNA polymerase –> RNA pol stalls/paused –> this causes transcription to be terminated
Rho-dependent termination
-Hairpin structure AND stretches of uracils are still present and utilized
-Rho protein plays a role in transcription termination
Rho binds to its utilization site in RNA
moves along the RNA until it reaches the RNA polymerase that is stalling/paused at the termination site
Rho protein has what type of activity?
Helicase activity. Rho will separate the RNA-DNA duplex, releasing RNA. Now, transcription is terminated.
Some antibiotics prevent
bacterial cell growth by inhibiting its RNA synthesis
Rifampin
an antibiotic used in treating tuberculosis
Rifampin mode of action
Binds to the beta subunit of prokaryotic RNA polymerase. The inhibited enzyme remains bound to the promoter, thereby blocking the initiation of RNA synthesis.
Rifampin does not bind to
eukaryotic RNA polymerase
Eukaryotic Transcription events (4)
- Initiation
- Elongation
- Termination
- Post-transcription processing
Initiation in eukaryotes
-Binding of RNApol and general transcription factors
-Formation of Pre-initiation complex (PIC)
Elongation in eukaryotes
-Conformational changes in RNApol through phosphorylation
-Start of transcription and unwinding, elongation, and re-annealing
Termination in eukaryotes
-Recognition of AAUAAA sequences
-Released of newly synthesized transcript (RNA)
Post-transcriptional processing
-5’ cap
-3’ poly A tail addition splicing
-Splicing
-RNA editing
Eukaryotes have 3 types of RNA polymerases that transcribe distinct classes of RNA (1 polymerase in prokaryotes)
- RNApolI (transcribes three major rRNAs)
- RNApolII (transcribe mRNA)
- RNApolIII (transcribes tRNA)
*1,2,3 = r,m,t
General Transcription Factors (GTFs)
initiate transcription (no GTFs in prokaryotes).
Nomenclature: if GTFs associated with RNA polymerase II, then the factors are termed “TFII,” for “Transcription Factor (involving) RNA polymerase II”
Eukaryotic Transcription Initiation (First three steps)
- TFIID: binds to AT rich promoter element called TATA box (TFIID, a multisubunit complex, contains TATA binding protein TBP). TBP allows TFIID complex to recognize and bind to the TATA box. (Note: TBP is analogous to bacterial sigma factor). TATA at approx -10 posiiton.
- TFIIA assits TFIID binding
- TFIIB orients the RNA polymerase
Eukaryotic Transcription Initiation (Steps 4, 5, and 6)
- RNA polymerase II is bound to the GTFs at the promoter. The carboxy-terminal domain (CTD) of RNA polymerase II is held in place by the GTFs.
- TFIIE interacts with promoter bound RNA polymerase II
- TFIIH is recruited
Pre-initiation complex (PIC)
All these TFII’s are required by RNApol II for transcription
TFIIH
a multi-subunit protein complex that has two activities that are important for initiation of transcription
-Helicase activity: unwinds DNA at the initiation site
-Kinase activity: phosphorylates the C-terminal domain (CTD) of RNApolII
Upon CTD being phosphorylated, RNApolII no longer
binds to GTFs and GTFs are released from the complex. Then elongation of transcription starts.
Transcription requires
NTPs (nucleoside triphosphates)
Termination sequence
AAUAAA
Gene transcription produces an RNA that is?
Larger than the mRNA found in the cytoplasm for translation. This is called the primary transcript.
Eukaryotic mRNA primary transcripts, or “pre-mRNA” undergo modifications to provide
Stability to the mRNA and to assist in its translation by ribosomes. This is referred to as post-transcriptional modifications (PTMs)
What are the four post-transcriptional modification processing evetns?
- 5’ capping
- Splicing
- 3’ polyadeylation
- RNA editing
What is the first processing reaction for pre-mRNA?
5’ capping with 7-methylguanosine
A 7-methylguanosine is attached to the 5’-terminal end of the mRNA when?
as soon as mRNA synthesis begins through a 5’–>5’-triphosphate linkage.
7-methylguanosine
is a guanosine with a methyl group (-CH3) attached at position 7 of the purine ring.
The addition to the 5’ end provides what?
Stability to the RNA molecule by protecting the transcript from degradation by nucleases
The 5’ cap also plays an important role in the
positioning of mRNA on the ribosome for initiation of translation
The primary transcripts contain a highly conserved….
AAUAAA consensus sequence known as the polyadenylation signal, near the 3’ end
A specific endonuclease recognizes the what sequences and cleaves what?
Recognizes the AAUAAA sequences and cleaves the primary transcript 20 nucleotides downstream from the AAUAAA sequence.
After the cut, enzyme poly(A) polymerase (aka, polyadenylate polymerase) adds what to where? (3’ cap)
Adds 20-450 adenylates (adenosine monophosphates) one at time to the 3’using ATP as the substrate. This creates a poly-A tail.
What terminates eukaryotic transcription?
PolyA tail
PolyA tail summary
-Required for the stability of the mRNA
-Required for efficient re-initiation of translation (interacts with 5’ cap)
-Functions as a signal for export of the mRNA from the nucleus to the cytoplasm
Maturation of eukaryotic mRNA usually involves
removal from the primary transcript of RNA sequences that do not code for protein, ie: removal of introns or intervening sequences
Exons
expressed region = protein coding sequences
Introns
intervening regions = noncoding sequences
Most eukaryotic genes contain
exons interrupted by introns
Mature mRNAs are formed when?
Intron sequences are removed and exons sequences are spliced (ligate) together all by spliceosome
Mature mRNAs have
exons but no introns and a 5’ and 3’ UTR sequences. UTRs are located within the first and last exons
Spliceosome converts the primary transcript into
mRNA
Each spliceosome is composed of?
Five small nuclear RNAs (snRNPs) along with additional proteins to mediate splicing
What do spliceosomes do?
Facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron.
Although the sequences within introns may differ widely, there are how many critical nucleotide sequences? They are always…?
Three critical nucleotide sequences that are always present in introns. These elements direct the removal of an intron. (GU, A, AG)
GU
site of splicing at the 5’ end of an intron
A
branch point of a lariat formation
AG
site of splicing at the 3’ end of an intron
RNA Splicing first two steps
- The 2’OH group of an A of the branch point attacks to the phosphate at the 5’ end of the intron, creating a “lariat” structure
- The newly freed 3’OH of exon 1 (G sequence at the 5’ donor site” attacks the 5’ phosphate at the splice acceptor site, forming a phosphodiester bond that joins exons 1 and 2
RNA splicing Steps #4 and #5
- The excised intron is released as a lariat which is degraded by nucleases
- After exons ligate, the mature mRNA molecules translocate into the cytosol
B-thalassemia
-Correct splicing is important: about 20% of inherited human disease involve splicing errors in pre-mRNA
-B-thalessemia is a hereditary anemia disease in which the production of the B-globin protein is defective
Cause of B-thalassemia
Single point mutation at the splice junction sequences at the intron-exon boundaries cause the incorrect splicing of B-globin mRNA (ex: GU to AU at the 5’ donor site)
Since most pre-mRNAs contain multiple introns, different mRNAs can be produced from the same gene by
different combinations of 5’ and 3’ spice sites.
- > 90% of human genes can be spliced in alternative ways in different tissues.
The use of different patterns of splicing to give rise to
different mRNA sequences
(and thus different protein products) is called alternative splicing.
Benefit of alternative splicing
producing a large diverse set of proteins from a limited set of genes
ex: isoforms of the tropomyosin protein (involved in muscle contraction)
mRNA editing
Process through which the nucleotide sequence specified in the mRNA is modified to produce a different nucleotide sequence (does not affect DNA).
RNA editing involves the modification of?
A single RNA nucleotide to alter sequence. Result: allowing the production of alternative protein products from a single gene
RNA editng is an important mechanism of
genetic regulation that amplifies genetic plasticity by allowing the production of alternative protein products from a single gene
mRNA editing occurs after
the transcript is synthesized, so the mature mRNA differs in different tissues
Example of RNA editing
cytosine to uracil (deamination reaction) in RNA
Apoprotein B gene (apoB)
is an essential component of chylomicrons (CMs) and very-low-density lipoproteins (VLDLs) (mRNA editing example)
ApoB and mRNA is made in the
liver and small intestine
In liver, cytidine is…
NOT present, mRNA is unedited and translated to YIELD ApoB-100.
In intestine, deaminase is…
PRESENT, mRNA is edited, cytosine is deaminated to uracil. This changes glutamine to a stop codon. This results in a shorter protein called apoB-48.
mRNA allows the
production of alternative protein products for a single gene
As the mRNA emerges from the polymerase it is….
it is capped, then as introns
emerge, they are removed, and the exons are spliced together. The poly A tail is added immediately after transcription terminates.
Pre-mRNAs are modified into
mature mRNAs while being synthesized. This is described as co-transcriptional mRNA processing.
In both prokaryotes and eukaryotes, gene promoters can be located on
either strand of DNA
The direction of transcription is determined by the
promoter at the beginning of each gene, which is recognized by RNA polymerase.
Since RNA polymerases always transcribe in the (what direction?) the direction of movement will be dependent on?
Since RNA polymerases always transcribe in the 5’ to 3’ direction, the direction of movement will be dependent on the strand the polymerase binds to and is
using as a template.
