Transcription and RNA processing Flashcards
On the ribose sugar of RNA, the 2’ carbon bears a ___________ _________
hydroxyl group
How can the structure and function of RNA resemble proteins
has tertiary structure
may interact as functional units (quaternary structure)
How is RNA unstable
the presence of the unique 2’ OH group in ribose causes it to react intramolecularly with the 3’ OH site resulting in phosphate bond breakage
Why use RNA if it is unstable?
since RNA is single stranded, it is able to take on many forms and has tertiary structure associated with it for many functions
What types of RNA are transcribed in both prokaryotes and eukaryotes
mRNA, rRNA, and tRNA
What types of RNA are only produced in prokaryotes
CRISPR RNA
What does it mean when saying transcription and translation are coupled
they occur simultaneously
Transcription in prokaryotes takes on a Christmas tree like structure; the longest strands would be the ______________ in age, and the shortest would be the ________________ in age
longest = oldest
shortest = newest
Why are ribonucleoside triphosphates used in RNA instead of deoxyribonucleoside triphosphates
not deoxy because its RNA - has the hydroxyl group
What is the RNA polymerase in prokaryotes
holoenzyme
What is the function of the alpha subunit of holoenzyme (there are 2 of these per holoenzyme)
assembly of tetrameric core
What is the function of the beta subunit of holoenzyme
contains ribonucleoside triphosphate binding site
What is the function of the beta prime subunit of holoenzyme
contains the DNA template binding region
What is the function of the omega subunit of holoenzyme
stabilizes tetrameric core
What is the function of the sigma subunit of holoenzyme
binds to the RNA polymerase tetrameric core and assists in the correct initiation of transcription (these allow for specificity)
What is the tetrameric core of holoenzyme comprised of
two alpha subunits, a beta subunit, and a beta prime subunit
Is transcription of RNA in prokaryotes sequence dependent or independent
sequence dependant
Is transcription of RNA in prokaryotes primer dependent or primer independent
primer independent
What are the important sequences needed for transcription of RNA in prokaryotes
-35 consensus (TTGACA)
-10 consensus (TATAAT)
What factor recognizes and binds to the -35 and -10 consensus sequences
sigma factor
Why is the -10 promoter sequence prone to unwinding
weak H-bonding (AT rich)
How does the initiation of RNA transcription occur in prokaryotes
- The sigma factor of holoenzyme first recognizes and binds to the consensus factors on the DNA sequence
- then RNA poly is positioned above the +1 site (start) and has unwound DNA to produce a single strand
- RNA poly binds, unwinds, and joins the first 2 nucleotides
Does RNA synthesis in prokaryotes require a primer
no
How does elongation of RNA transcription occur in prokaryotes
- holoenzyme binds tightly and unwinds the double strand
- rNTP complementary to the first base pair on +1 site serves as the first nucleotide
- two phosphate groups are cleaved from the subsequent rNTP, creating a nucleotide that is added to the 3’ end of the growing RNA molecule
- sigma factor is released as RNA poly moves beyond the promoter
- complementary bases are continually added
How does termination of RNA transcription occur in prokaryotes
- RNA poly reaches a terminator region, but this occurs upstream of where termination actually takes place
- newly synthesized RNA as well as RNA poly are released
What are the two terminators possessed by bacterial cells
Rho-dependant (requires Rho) and Rho-independant (intrinsic terminator)
What are the 2 sequence features of Rho dependant termination
- sequence causes polymerase to pause
- DNA sequence upstream of terminator encodes a stretch of RNA that is C rich
What is the C rich sequence upstream of the Rho dependant termination site called
the rut site
Where does Rho bind in Rho dependant termination
the rut site
What activity does Rho factor have
helicase activity (unwinds the DNA RNA hybrid)
- ends transcription
What are the 2 features of Rho independent termination
- contains inverted repeats
- string of 6-9 A’s follows the inverted repeats
What occurs in Rho independent termination
the poly A sequence is transcribed into a poly U tail after the hairpin is transcribed which causes polymerase to pause
- the hairpin forms an destabilizes the DNA/RNA hybrid
(assisted by weak A U pairing)
What is the purpose of “puffs” (balbani rings) in eukaryotic transcription
make a lot of protein/enzyme
What are the most important polymerases to know in eukaryotic transcription
poly 1 2 and 3
What is the main polymerase for making proteins in RNA
poly 2
Transcription in eukaryotes is still sequence dependant and primer independent, but what is different about initiation of eukaryotic transcription
requires transcription factors
How is eukaryotic transcription initiation specific
accessory proteins recognize specific promoters (specific to each protein) and notify the appropriate polymerase (ie. transcription initiation is dependant on the promoter sequence and the accompanying accessory proteins)
What makes promoters on eukaryotes more complex
has a core promoter and a regulatory promoter
Instead of sigma factor, what completes the step involving initiation in eukaryotes
polymerase 2
What does polymerase 2 create in the initiation of eukaryotic transcription
transcription factors of poly 2 (TFII)
What allows assembly of the TATA box in relation to poly 2
TATA-binding protein (TBP)
What is the preinitiation complex (PIC)
the complex that initiates transcription in eukaryotes (poly 2, TFIIs, TBP)
What is the more complex transcriptional regulation complex that permits interactions with other activator/repressor proteins
mediator
What is meant by the term basal transcriptional machinery
the components required for promoter interaction in eukaryotic transcription
Why does the DNA strand loop around in the initiation of eukaryotic transcription
so the transcription factors bound to enhancing sequences can interact with the basal transcription apparatus
Why does the transcription factor stay bound to the DNA as DNA poly synthesizes RNA in eukaryotic transcription
so new initiation can occur at the promoter
How long is the transcription bubble in the elongation stage of eukaryotic transcription
8 nucleotides
What is the function of RNA poly 1 in eukaryotic transcription
requires a termination factor (similar to Rho in prokaryotes)
What is the function of RNA poly 3 termination in eukaryotic transcription
transcribes a terminator sequence of U’s downstream of a hairpin
What is the function of RNA poly 2 in eukaryotic transcription termination
continues transcribing past the termination sequence (RNA is cleaved at a consensus sequence in the RNA)
How does termination in eukaryotic RNA transcription work
cleavage occurs at the 3’ coding region in the transcribed RNA, Rat1 exonuclease attaches to the 5’ end of trailing RNA and degrades it as it moves toward RNA poly, once it reaches RNA poly transcription is terminated
What is the main difference between Rho and Rat1
Rho binds and doesn’t chew up
Rat1 binds and chews up
What is the purpose of the regulatory promoter region in eukaryotic RNA transcription
binds transcription factors that positively or negatively influence transcription
What type of RNA does RNA poly 2 transcribe in eukaryotes
mRNA
What type of RNA does RNA poly 1 transcribe in eukaryotes
large RNAs
What type of RNA does RNA poly 3 transcribe in eukaryotes
rRNAs and tRNAs
What is colinearity when referring to RNA
the sequence of the gene directly determines the amino acid sequence - they are collinear
What sequence is seen in the RNA processing of prokaryotic RNA
Shine Dalgarno sequence
What is the 5’ untranslated region (5’UTR)
before the start codon, doesn’t code for amino acids but binds to the ribosomal complex
What is the protein coding region
comprises of codons that code for specific amino acids (begins w start codon and ends w stop codon)
What is the 3’UTR
does not code for amino acids, affects the stability of the mRNA and regulates its translation
What does it mean when saying in eukaryotes gene expression is often ‘interrupted’
exons (protein coding) are often accompanied by introns (non coding)
What must happen with introns in a sequence
must be removed
Where does splicing of introns occur in the cell
the nucleus
What is required for splicing
spliceosomal complex that contains smaller nuclear RNAs (snRNAs) and proteins
What are the three main processing steps in eukaryotic nuclear pre-mRNA
- addition of 7 methyl guanosine cap (5’ cap)
- addition of poly A tail
- removal of introns
Where are methyl groups added to form the 5’ cap
- 7’ position of the terminal guanine
- 2’ OH groups of the sugar of the first (& sometimes second) nucleotide
- to adenine if it is the second nucleotide (sometimes)
What is the formation of the poly A tail
50-250 A’s added to the 3’ end (polyadenylation)
- polyadenylation signalled by the poly A consensus sequence 11 to 30 nucleotides upstream (AAUAAA)
What is the poly A consensus sequence that signals polyadenylation
AAUAAA
What does the addition of poly A tail facilitate (think movement)
export out of the nucleus
What is RNA splicing
removal of introns from pre-mRNA
Every intron has ___ conserved sequences that are required for precise removal
3
What are the 3 conserved sequences required for precise intron removal
5’ splice site, 3’ splice site, an branch point
What are the steps of RNA splicing
- pre-mRNA is cut at 5’ splice site and the 5’ end of the intron attaches to branch point
- cut is made at 3’ splice site and simultaneously the 3’ end of exon 1 attaches to 5’ end of exon 2
- intron is released as a lariat and degraded
What is a spliceosome
an RNA/protein structure that contains five small nuclear RNAs (snRNAs) designated U1, U2, U4, U5 and U6
- these snRNAs associate with about 300 small proteins to form small nuclear ribonucleoproteins (snRPSs)
Where does U1 attach
5’ splice site
Where does U2 attach
branch point
Where do U4 U5 and U6 go
form a complex and join the spliceosome
What is the function of the U4 U5 and U6 complex
conformational change in the sequence that folds it and brings the 5’ end close to the branch point
What happens after U4 U5 and U6 complex folds 5’ end to the branch point
U1 and U4 are released, and base pairing occurs between U2 and U6, as well as between U6 and the 5’ splice site (now all in close proximity)
- exons are joined together and the intron is released as a lariat
What is alternative splicing
- pre-mRNA is processed in different ways to produce alternative types of mRNA
- alternative exons are used
- results in different proteins from the same DNA sequence
in essence - all exons can be included or only some, resulting in different products depending on what is included
What occurs in multiple 3’ cleavage sites
can generate a shorter or longer exon
occurs at the last exon
could produce a different protein
(*not the same as 3’ splice site - this is the U rich cleavage site previously covered)
in essence - exons have multiple cleavage sites so they vary in length and create different products as a result
How is alternate splicing and multiple 3’ splice sites important
increases diversity of proteins created
What is a real life application of alternate splicing
different tissues of the body produce different variations of exon strands to give rise to variability in the proteins created
How often do genes go through alternate splicing
95%
How often do genes have multiple 3’ cleavage sites
50%
What is RNA editing
information in the RNA strand is altered
What are gRNAs (think RNA editing)
guide RNAs - direct insertion of uridine bases in mRNA (by a repair polymerase)
What does the function of gRNAs result in
permanently modifies the mRNA by making new codons that specify new amino acids in the protein
What are apoplipoproteins
blood proteins that carry lipids
What enzyme contributes to RNA editing changing C to U
cytidine deaminase
What is the point of cytidine deaminase
convert a normal glutamine codon (CAA) to a termination codon (UAA) which ends the protein and gives rise to a different function
What are the steps of tRNA modification
- precursor tRNA is cleaved to produce just the individual tRNA molecule
- introns are removed via splicing
- bases are added to the 3’ end
- modification of several bases produce mature tRNA
What enzymes contributes to modifying tRNA
tRNA modifying enzymes
What is different about the removal of introns in tRNA
no spliceosomes required
What makes up a ribosome
a large and small subunit
What are the protein amounts associated with prokaryotic ribosomes
large subunit: 50S
small subunit: 30S
(come together to form 70S)
What are the protein amounts associated with eukaryotic ribosomes
large subunit: 60S
small subunit: 40S
(come together to form 80S)
How many genes and products in prokaryotic ribosomes
1 gene: 3 products (16S, 23S, and 5S)
How many genes and products in eukaryotic ribosomes
2 genes (large and small on separate): 4 products (18S, 5.85S, 28S on large, 5S on small)
Where is the site of rRNA synthesis in prokaryotes
cytoplasm (no nuclear envelope)
Where is the site of rRNA synthesis in eukaryotes
nucleolus
What are snRNAs
small nuclear RNAs - spliceosomes
What are snoRNAs
small nucleolar RNAs - ribosomal
What is the role of snRNAs (spliceosomes)
play roles in post-transcriptional processing (ie. splicing)
What is the role of snoRNAs (ribosomal)
in eukaryotes, guide enzymatic modifications of ribosomal RNAs, transfer RNAs, and small nuclear RN
What are siRNA and miRNA
found in eukaryotes and act as short complementary sequences that bind to complementary sequences in mRNA
- control gene expression in various ways
What is CRISPR RNA
found in prokaryotes and works with Cas9 to cleave foreign DNA that enters a host cell (bacterial defense system)
What are LncRNAs
long non-coding RNAs
- function in eukaryotic cells to regulate and control gene expression at the level of transcription of translation
OR
- bind and recruit proteins involved in DNA modification
What initially binds to the TATA box on the DNA template in eukaryotes
TFIID
When eukaryotic mRNA is hybridized to the complementary DNA, loops of unhybridized DNA are seen, what do they mean
they correspond to non coding regions of the gene and demonstrate that genes and proteins are not collinear
