Transcription Flashcards
Transcription
DNA directed synthesis of RNA
Transcription is the first step of…
Gene Expression
RNA is transcribed from…
only ONE strand of DNA from a gene
Template Strand
The strand that serves as a template for complementary RNA transcript formation
Template strand is also known as:
1) Non-coding strand
2) Anti-sense strand
Why is the template strand also called the “non-coding strand”?
Because its sequence is not the same as the RNA produced from it (it has a complementary sequence)
Non-Template Strand
AKA –> Coding Strand / Sense Strand
–> The strand that is NOT transcribed
Why is the non-template strand also called the “coding strand”?
Because its sequence is “the same” as that of the RNA transcribed from the template strand
–> Both the non-template strand and RNA transcript are complementary to the template strand
Every time a gene is transcribed, the ________________ is always used as the template
SAME STRAND
Why is the same strand of a gene always used as the template during every round of transcription?
Particular DNA sequences associated with a gene determines how RNA polymerase is oriented when it binds to the DNA which then established which strand will be utilized as the template
–> This DOES NOT mean the same strand is used for ALL genes: it is gene specific which strand is the template
Genes are READ: ______________
So the RNA transcript is produced: ______________
Genes are read 3’ —-> 5’
RNA transcript is produced 5’ —–> 3’
Differences between DNA and RNA (3)
1) RNA uses Uracil instead of Thymine
2) RNA is usually single stranded
3) RNA contains ribose instead of deoxyribose
How does RNA being single stranded affect its stability?
Makes it less stable than DNA
–> It is more vulnerable to degradation/digestion by nucleases
How does ribose affect the stability of RNA?
Makes it less stable
–> Under basic conditions the hydroxyl group on 2’ C may be deprotonated which can act as a neucleophile and hydrolyze (break) the phosphodiester bonds between nucleotides
Importance of stability to RNA and DNA:
1) DNA is chemically stable: Important because DNA is PERMANENT –> The cell does NOT want the DNA to be changed
2) RNA is chemically unstable: Important because RNA needs to be short-lived/temporary so that the cell can control its quantity and therefore the amount of protein produced
–> cell needs to be able to degrade RNA to control protein levels
Major Cellular RNAs
1) mRNA –> Messenger RNA
2) tRNA –> Transfer RNA
3) rRNA –> Ribosomal RNA
4) snRNA –> Small Nuclear RNA
5)ncRNA –> Non-Coding RNA
mRNA
Messenger RNA
–> Encodes for proteins (what ends up getting translated)
tRNA
Transfer RNA
–> Functions during translation in which it serves as an “adaptor” molecule to bring the correct amino acid to the translation machinery (ribosome)
rRNA
Ribosomal RNA
–> Functions during translation in the structure of ribosomes
–> AND certain rRNAs are involved in the CATALYSIS of peptide bond formation
What is the most abundant RNA?
rRNA
snRNA
Small Nuclear RNA
–> Functions during RNA splicing
Structural RNAs
RNAs that are encoded by genes but DO NOT get translated into proteins:
1) tRNAs
2) rRNAs
3) snRNAs
–> Made of RNA but function kind of like proteins
ncRNA
Non-Coding RNA
–> We got no clue what they do: preliminary studies have found some evidence they may be involved in gene expression regulation
RNA Polymerase
An enzyme that uses a single stranded DNA template to synthesize a complementary strand of RNA
RNA polymerase builds RNA strand in the…
5’ to 3’ direction
–> Adds nucleotides to the free 3’ end of the synthesizing strand
(Like DNA polymerase in this way)
RNA polymerase also has ____________ activity
HELICASE ACTIVITY
–> Unwinds the DNA by itself to free the template strand (no other enzyme needed to separate the DNA strands)
RNA polymerase DOES NOT NEED…
a PRIMER –> Able to start a nucleotide chain from scratch
(unlike DNA polymerase)
Similarities between RNA and DNA Polymerases: (2)
1) Template-directed nucleic acid polymerization
2) Works in the 5’ to 3’ direction, adding nucleotides to the free 3’ end of the new strand
Differences between DNA and RNA production: (7)
1) DNA uses ATGC and RNA uses AUGC
2) DNA uses dNTPs and RNA uses NTP (nucleotide triphosphates)
3) DNA stays in nucleus and RNA can leave
4) DNA replication = double stranded molecule
RNA transcription = single stranded transcript
5) DNA rep. = ALL of genome is copied
RNA transcrip. = copies only a SEGMENT of DNA
6) DNA Polymerase needs primer and RNA polymerase does NOT
7) DNA rep. = CAREFUL PROOFREADING
RNA transcrip. = Very minimal to no proofreading
What is the reason for difference in proofreading between DNA and RNA?
DNA –> Needs extensive proofreading as any errors left in the code would be inherited causing incorrect genetic code to proliferate
RNA –> Doesn’t matter as much; so much RNA is produced that a few errors here or there wont have a huge impact as any faulty proteins produced would just get degraded (not a permanent problem if there is an error in the code)
RNA Polymerase in Prokaryotes vs Eukaryotes
Prokaryotes = Only have ONE RNA polymerase that produces ALL RNA molecules
Eukaryotes = Have THREE RNA polymerases
1) RNA Polymerase I
2) RNA Polymerase II
3) RNA Polymerase III
RNA Polymerase I
Synthesizes 3 rRNAs
RNA Polymerase III
Synthesizes tRNA + 1 rRNA
(Think Three = Transfer) –> The T’s match
RNA Polymerase II
The main one!
–> Synthesizes pre-mRNAs AND snRNAs
In-Vitro Transcription
Put into a test tube:
1) DNA
2) RNA Polymerase
3) NTPs (ATP, GTP, CTP, UTP)
4) Mg2+ (a cofactor of RNA polymerase)
–> RNA transcription will occur RANDOMLY –> Non-specific transcription
How does in-vivo transcription differ from in-vitro?
In-vivo is SPECIFIC –> Highly regulated
In-vitro is NON-SPECIFIC –> Not regulated and random
What regulates in-vivo transcription?
1) Recruitment of RNA polymerase
2) Transcription factors
3 main stages of transcription:
1) Initiation
2) Elongation
3) Termination
Transcription Initiation (General overview)
The recruitment of RNA polymerase and binding of it to the promoter leading to the unwinding of DNA and beginning of RNA synthesis
Promoter
Region of the gene that contains the start site of transcription and regulates efficiency of transcription
–> DNA sequences that define the start site of transcription
Where are promoters located?
Adjacent to the DNA they regulate (Upstream of the coding region of a gene)
Transcription Start Site (TSS)
+1 Position
–> Within the promoter and where transcription actually starts
Upstream of the TSS is…
A non-transcribed region of the promoter where RNA polymerase attaches
Cis-Acting Elements
“On the same side as”
Cis-acting DNA promoter elements
The non-transcribed regions of the promoter that regulate transcription by acting as binding sites for DNA binding proteins that regulate initiation
Consensus Sequences
A sequence of DNA having similar structure and function in different organisms
Transcription Unit
Stretch of DNA DOWNSTREAM from the TSS that gets transcribed into RNA
2 methods of initiation in prokaryotes and eukaryotes:
1) Prokaryotes –> RNA polymerase DIRECTLY binds to the promoter
2) Eukaryotes –> RNA polymerase requires additional transcription factors for its recruitment and binding to the promoter
Transcription Factors (TFs)
Proteins that recognize and bind to specific DNA sequences within the promoter region
–> When TFs are bound to the promoter, RNA polymerase is recruited allowing for it to bind to the promoter
What determines the direction of transcription?
Interactions between TFs and RNA Polymerase determine the precise location and orientation in which RNA polymerase will bind to the promoter
–> This binding then determines where transcription starts (on what strand) AND THEREFORE the direction it will proceed in
Transcription Initiation Complex
== (Assembly + binding of TFs to promoter) +
(RNA Polym. II Bound to promoter)
RNA polymerase begins transcription once…
the transcription initiation complex is formed
Initiation Process in Eukaryotes:
1) Several TFs bind to the promoter region
–> One TF must bind to the TATA box
2) RNA Polymerase II is recruited
3) Additional TFs and RNA Polymerase II bind to the DNA
= Initiation Complex Formed
–> Transcription begins!
What determines the rate of transcription?
The “strength” of the promoter
Rate of Transcription
= # of RNA molecules transcribed by a given template
Strong vs Weak Promoters
Strong Promoters = High binding affinity of RNA polymerase and TFs to the promoter –> Greater rate of transcription (more mRNA)
Weak Promoters = Lower binding affinity of RNA polymerase and TFs to the promoter –> Lower rate of transcription (less mRNA)
Affinity + Binding Affinity
Strength of the attraction between 2 substances
–> High binding affinity = tighter binding between two substances
What determines the binding affinity of promoters to TFs and RNA polymerase?
The promoter sequence
Elongation
After transcription is initiated, RNA polymerase moves along the DNA and untwists it (exposing 10-20 nucleotides at a time) to produce a single stranded template
–> RNA polymerase adds nucleotides to the 3’ end of the new growing RNA strand
What enzyme functions downstream of RNA polymerase?
Topoisomerase –> Relieving supercoiling
As RNA polymerase chugs along, the region behind RNA polymerase…
closes up and REWINDS into a double helix
(the section that rewinds is upstream of RNA polymerase: towards the 3’ direction of the template strand)
What allows for gene expression amplification in the process of transcription?
By the simultaneous transcription of a single gene
–> After one RNA polymerase begins transcription and goes on its way, another RNA polymerase can bind to the promoter of the same gene and begin transcription following behind the polymerase in front of it
–> Increases the amount of mRNA transcribed and therefore increases the amount of protein produced
The simultaneous transcription of a gene allows for…
the cell to make encoded proteins in LARGE amounts (easier to meet demands)
Termination
The stopping of transcription
–> 2 different methods between prokaryotes and eukaryotes
Termination in Prokaryotes
1) RNA polymerase stops transcription right at the end of the terminator
2) RNA and DNA are released from RNA Polymerase
3) The RNA released is IMMEDIATELY ready to undergo transcription (no additional processing)
Termination in Eukaryotes
1) The polyadenylation sequence is reached (AAUAAA)
1.1) Transcription continues on past this signal
2) pre-mRNA is then immediately bound by proteins in the nucleus which then cut the pre-mRNA free around 10-35 nucleotides downstream of the AAUAAA sequence
Polyadenylation Sequence
AAUAAA –> The “Cleavage Signal”
–> Signals for transcription to end 10-35 nucleotides downstream of the AAUAAA sequence
pre-mRNA
Immature mRNA = PRIMARY TRANSCRIPT
–> Must undergo processing BEFORE it can leave the nucleus to be translated
Post-Transcriptional RNA Modification
Enzymes in the nucleus modify pre-mRNA BEFORE it is transported to the cytoplasm for translation
RNA Processing
The sequence of events through which the primary transcript of a gene acquires its mature form
(getting pre-mRNA to mRNA)
3 main RNA modifications:
1) 5’ mG Cap
2) 3’ Poly-A Tail
3) RNA Splicing
Post-Transcription Modification Regulates: (3)
1) Stability of RNA
2) Transport out of the nucleus
3) The sequence of the RNA itself –> Affecting the final protein produced
5’ mG Cap
5’ methylguanosine cap
–> Modified form of guanine nucleotide is added to the 5’ end of the pre-mRNA
Modified guanine in the 5’ Cap
Guanine nucleotide + methyl group on the 7 carbon
–> Connects to the 5’ end of pre-mRNA with a TRIPHOSPHATE LINKAGE
Purpose of 5’ Cap
1) Helps to protect mRNA from exonucleases (prevents degradation)
2) Later on it functions as an “attach here” signal for ribosomes (helps ribosomes connect to the right side for proper start of translation)
3’ poly-A Tail
At the 3’ end, 50-250 adenine nucleotides are added
–> After the polyadenylation signal, proteins cleave the pre-mRNA and then add on the poly-A tail
What enzyme adds the poly-A tail?
Poly (A) Polymerase
–> Uses ATP to add adenine which leaves behind (PPi)
Purpose of Poly-A Tail
1) Facilitates EXPORT of mRNA from the nucleus to the cytoplasm
–> Stabilizes the mRNA
2) Helps to protect the mRNA from nuclease degradation
Final mRNA
5’ Cap –> 5’ UTR –> START Signal –> Spliced Exons –> STOP Signal –> Polyadenylation signal (AAUAAA) = 3’ UTR –> 3’ Poly-A Tail
Function of UTRs
UTR = Untranslated region
–> Functions in aiding with ribosome binding
RNA Splicing
The removal of non-coding segments which lie between coding segments of pre-mRNA and then the subsequent splicing (joining) together of the coding segments
–>Removal of introns + splicing of exons
Introns
Non-Coding Segments –> “Junk DNA”
–> Gets removed (doesn’t get translated)
Intron = Interference
Exons
Coding Segments
–> Gets joined together and STAYS (gets translated)
Exon = Expressed
The final mRNA transcript contains…
and is therefore…..
EXONS spliced together
–> It is therefore MISSING bits of the gene encoded by the original DNA
Spliced RNA and their template DNA are NOT __________
CO-LINEAR
TheDNA that encodes for polypeptides is NOT ___________ but is instead separated into ______________
1) Continuous
2) Segments
Where/when does RNA splicing occur?
Occurs in the nucleus BEFORE the mRNA leaves to the cytoplasm
Spliceosome
The splicing machinery
–> A large ribonucleoprotein complex =
composed of many proteins and snRNPs (5)
snRNP
small nuclear ribonucleoprotein
= Several proteins + snRNAs
Steps of Splicing:
1) snRNPs (5) + other proteins combine and bind to the pre-mRNA forming a spliceosome
2) Within the spliceosome, the snRNA part of the snRNPs recognize and base pair to the sequences at the ends of an intron (the junctions between exons and introns)
3) Binding to the ends of the intron causes it to fold inward, bringing the 5’ and 3’ end of the intron towards each other and forming a loop (LARIAT)
4) Separated exon ends move towards each other and the pre-mRNA is cut at the intron-exon junctions
5) Adjacent exons get spliced together and the intron is released
6) Spliceosome comes apart –> releases the spliced mRNA containing only exons now
Splicing is catalyzed by…
the RNA component of the spliceosome (snRNA)
Ribozymes
RNA molecules that function as enzymes
–> Their discovery invalidated the idea that all biological catalysts are proteins
Purpose of RNA Splicing
We don’t know for sure why splicing exists (as it seems like a wasteful system)
HOWEVER:
It does allow for the information in DNA to be used in many different ways
Alternative RNA Splicing
A process in which exons from the same gene are joined in different combinations to form different but related mRNA transcripts
–> Enables for one gene to encode for more than one polypeptide depending on which segments are treated as exons during splicing!
Alternative RNA splicing generates…
Protein diversity
Because of alternative RNA splicing, the # of proteins produced…
is much greater than the # of genes we have
Exon Shuffling
–> What does exon shuffling lead to?
Several exons of a gene encode for different domains (subunits) of the same protein and so alternative splicing allows for these exons to be shuffled and produce variants of the same protein with different domains
–> Leads to evolution of new proteins
–> Leads to the production of different variants of a protein to meet the functional requirements of specialized cells
EX: Alpha-tropomyosin
Transcription to Translation in Prokaryotes
Transcription and translation occur SIMULATANEOUSLY
–> All occurs within the cytoplasm: NOT compartmentalized
Transcription to Translation in Eukaryotes
Transcription and translation are compartmentalized
Nucleus = Transcription Machinery
Cytoplasm = Translation Machinery
Polycistronic mRNA
An mRNA that encodes for SEVERAL proteins
–> a single mRNA contains the info from multiple genes
–> Commonly found in prokaryotes but NOT in eukaryotes
UTR
Untranslated Region
–> IS FOUND IN THE mRNA: NOT IN THE DNA!
–> Includes things like the promoter, 5’ cap, etc.
Both strands of DNA can encode genes BUT…
The coding sequence of a gene will always be on ONE strand (the same one each time)
