Chapter 26: RNA Metabolism Flashcards
DNA-dependent synthesis of RNA (process of copying a piece of DNA (a gene) as RNA)
Transcription
Which DNA strand is the template strand for transcription?
non-coding strand
Which Ribonucleic Acid encodes amino acid sequences of all the polypeptides found in the cell?
Messenger RNAs (mRNA)
Ribonucleic Acids?
Messenger RNAs (mRNA) Transfer RNAs (tRNA) Ribosomal RNAs (rRNA)
post-transcriptionally regulate the expression of genes, by binding to mRNA nucleotide sequences
miRNA
Transfer RNAs (tRNA)
During protein synthesis, match specific amino acids to triplet codons in mRNA
RNA component of the ribosome, interact with tRNA during translation
Ribosomal RNAs (rRNA)
Less understood RNA function in eurkaryotes?
micro RNA (miRNA)
Four ribonucleotide 5’-triphosphates
ATP, UTP, GTP, CTP
DNA-dependent RNA polymerase requires:
- DNA template, all
four ribonucleotide 5’-triphosphates (ATP, UTP, GTP, CTP)
-Mg2+
-*In transcription, UTP is used in place of TTP.
RNA polymerase elongates an RNA strand in what direction?
5’ to 3’ direction
3’ hydroxyl attack the alpha-phosphorous atom of the incoming nucleotide and release PPi.
RNA Polymerase elongation mechanism
What is “NTP”?
nucleoside triphosphate (NTP) molecule containing a nucleoside bound to 3 phosphate groups.
The DNA strand that serves as the template for RNA synthesis
Template strand (DNA non-coding region)
TRUE/FALSE:
DNA non-template (coding) strand sequence as the newly made RNA molecule (RNA transcript)
TRUE
RNA-DNA duplex of approximately 8 bp are created during elongation
After elongation, RNA peels away allowing the DNA duplex to reform.
A transcription bubble forms with about 17 bp of DNA unwound.
Polymerization
Large complex with 5 sub-units, alpha, alpha, Beta, Beta prime, and omega (core units), and a sixth subunit , sigma.
E. coli RNA polymerase
In E.Coli RNA Polymerase, the assembly and binding to UP elements (sequence upstream of promoter)
Two alpha subunits function
In E.Coli RNA Polymerase, main catalytic subunit in the large complex
Beta subunit
In E.Coli RNA Polymerase, responsible for DNA-binding in the large complex
Beta prime subunit
In E. Coli RNA Polymerase, protect the
polymerase from denaturation (structural but no catalytic function) in the large complex.
Omega subunit
In E. Coli RNA Polymerase, directs enzyme to the promoter (“the brains”) in the large complex.
Sigma subunit
What subunit directs the core complex to specific binding sites on the DNA and released from core complex once transcription started?
Sigma subunit
Why aren’t mistakes in RNA synthesis are
generally not critical?
many RNA copies are made from a single gene and they are rapidly degraded and replaced.
Which phase of transcription is:
RNA polymerase binds to specific sequences of DNA known as promoters.
Intiation
RNA polymerase binds to how many DNA nucleotides,?
about 100
70 bp upstream of the start site has what numbers?
Designated by negative numbers
30 bp downstream of start site has what numbers?
Designated by positive numbers
In Transcription Initiation, two important promoter regions?
-10 position, -35 position
The Pribnow box, or TATA sequence, is found at what position?
-10 position
The UP element is located at what position?
-40 to -60 position
3 AT rich regions in Initiation?
-10
-35
UP element
Transcript Initiation
Polymerase binds to the promoter (forms closed complex)
DNA is partially unwound near the -10 sequence (forming the open complex)
Transcription is initiated and the complex is converted into a conformationally distinct elongation form.
complex then moves away from the promoter, releasing the sigma subunit
Examples of controlling Transcription
Differences in promoter sequences, requiring several sigma proteins with different sequence binding preferences.
Accessory proteins can either be _____ or ______ of transcription (transcription factors)
Activators, Repressors
Which Transcription step?
- RNA chain elongation occurs in the 5’ to 3’ direction
- RNA polymerase moves the transcription bubble along the DNA strand, creating positive supercoiling ahead of transcription and negative supercoiling behind.
-Entirely processive (Cannot let go of
DNA until it has finished making the complete RNA
transcript)
Elongation
Require a pausing of RNA polymerase on the
DNA transcript, which allows time for
termination to occur.
Transcript Termination
What are :
Rho-independent termination
Rho-dependent termination
Types of Transcript Termination
What type of transcript termination?
- Self-complementary DNA sequence of RNA near the end of the transcript is usually followed by a string of three A residues (transcribed into
uridylates) at the 3’ end of the RNA strand. - Forms a stable hairpin structure.
- The polymerase pauses at this point,
possibly because of the “hairpin” (affects
associations between the RNA
polymerase and the RNA transcript)
Rho-independent
What type of transcript termination?
- Sequence of repeated A residues is missing but there’s usually a CA-rich region. The polymerase pauses at this site.
- The Rho protein binds to RNA at specific binding sites and migrates in the 5’ to 3’ direction until it catches up with the polymerase.
- The Rho protein can only catch up to the polymerase if it is paused on the RNA transcript, hairpin structure also occurs in the RNA transcript in this form of termination.
Rho-dependent termination
TRUE/FALSE:
The Rho protein is a helicase, and uses ATP hydrolysis to migrate along the RNA strand.
TRUE
Eukaryotes have ____ types of RNA polymerases which form distinct complexes, although they do share some subunits.
3
Which Eukaryotic RNA Polymerase?
Synthesizes mRNA precursors. Can recognize
thousands of promoters of varying sequence, through associated proteins.
RNA Polymerase II
Eukaryotic RNA Polymerases have how many subunits?
at least 12.
{Requires an additional array of transcription factors (accessory proteins) to form an active transcription
complex.}
Which subunit of Eukaryotic RNA Polymerase is similar to a Prokaryote RNA Polymerase subunit?
Eukaryote’s RBP1 subunit is similiar to bacteria’s Beta prime subunit
Why are extra subunits required in eukaryotic transcription?
Because the packaging of DNA is far more complex in eukaryotes
What is contained in RBP1 subunit long tail?
repeats of a “–YSPTSPS-“ sequence that
is important for regulation.
Transcription from RNA polymerase II
can be divided into 4 steps:
– assembly
– initiation
– elongation
– termination
Which step of Eukaryotic Transcription?
TATA-binding protein (TBP) binds to the TATA box. TFIIB, TFIIF-RNA Polymerase II, TFIIE, and TFIIH are recruited
Assembly
Eukaryotic Transcription Initiation factor:
Binds tightly to RNA PolI and guides it to the correct DNA sequences (complex)
Binds to TFIIB.
TFIIF
Binding of what completes the closed complex and starts to unwind the DNA to create the open complex?
TFIIH
Which step of Eukaryotic Transcription?
TFIIH is also responsible for phosphorylating
RNA Pol II numerous times in its C-terminal
domain, causing a structural change and
Initiation
What’s released as RNA Pol II synthesizes the first 60-70 nucleotides and enters the elongation phase?
TFIIE and TFIIH
Which step of Eukaryotic Transcription?
Dephosphorylation of RNA Pol II
Termination
Eukaryotic Transcription Initiation factor:
recognizes the TATA box
TBP (TATA-binding protein)
Eukaryotic Transcription Initiation factor:
stabilizes the binding of TFIIB and TBP
TFIIA
Eukaryotic Transcription Initiation factor:
Binds to TBP
Recruits TFIIF-Pol II complex
TFIIB
Eukaryotic Transcription Initiation Factor:
Recruits TFIIH; has ATPase and helicase activties
TFIIE
Eukaryotic Transcription Initiation factor:
Unwinds DNA at promoter (helicase activity); Phosphorylates Pol II I(within CTD); recruits nucleotide-excision repair
TFIIH
Eukaryotic RNA molecules are called what?
Primary transcripts
Eukaryotic transcripts generally contain information for how many genes?
A single gene
Eukaryotic transcripts have intervening non-coding sequences that have to be what?
spliced out of the RNA transcrip
In post-transcriptional processing, a cap consisting of a ________ residue is added to the _____ end of mRNA transcripts. The ___ end has to be cleaved and a poly(A) tail of 80-250 nucleotides is added.
7-methylguanosine , 5’, 3’
In Capping of the 5’ end, what is added to the first and possibly the second nucleotide of the mRNA?
methyl group
help protect the mRNA from ribonucleases and participates in binding to the ribosome.
The Cap
When is the “cap” added and where does it come from?
early in transcription and comes from a molecule of GTP
Is the “cap” methylated?
Yes. Cap is methylated at N-7 after it has been
added to the mRNA.
TRUE/FALSE:
Cap is bound to the polymerase complex by
the cap-binding complex.
TRUE
Capping likely marks the completion of what?
RNAP II’s switch from transcription initiation to elongation.
Enzymes involved in the Cap-Synthesizing complex of process for “Capping the 5’ End”?
- Phosphohydrolase
- Guanylyltransferase
- Guanine-7-methyltransferase
- 2’ O-Methyltransferase
TRUE/FALSE:
Mature eukaryotic mRNAs have poly(A)
tails of 80-250 nucleotides appended to their 3’ ends
TRUE
TRUE/FALSE:
Poly A tails protect eukaryotic mRNAs (also interacts with ribosome), but they cause degradation of bacterial
mRNAs.
TRUE
Endonuclease
Trims the Eukaryotic mRNA molecule back to within ~20 nucleotides
TRUE/FALSE:
The poly(A) tail shortens over time and transcripts that lack poly(A) tails are degraded in <30 minutes.
TRUE
Coding part of a gene
Exon
Non-coding part of a gene
Introns
TRUE/FALSE:
Introns must be removed from the final
mRNA product before it is transported to the cytosol
TRUE
- Are self splicing
- Interrupt mRNA, tRNA and rRNA genes
- Found within nuclear, mitochondrial, and chloroplast genomes
- Common in fungi, algae, and plants, also found in bacteria
Group I and II Introns
- Spliced by spliceosomes
- Most common introns
- Frequent in protein-coding regions of eukaryotic genomes
Splicesomal introns
- Spliced by protein-based enzymes
- Found in certain tRNAs in eukaryotes and archae
- Primary transcript cleaved by endonuclease
- Exons are joined by ATP-dependent ligase
tRNA introns
TRUE/FALSE:
Poly(A) site is ~15 to 20 nucleotides past a conserved AAUAAA sequence
TRUE
Group I Intron
Use guanylate (GMP, GDP, or GTP) to initiate the splicing process.
Nucleotide attacks the 3’-end of the first exon, generating a 3’-OH that will then attack at the 5’-end of the second exon.
3’ OH of a specific guanosine in the intron acts as nucleophile attacking phosphate at the 5’ splice site to form lariat structure
Intron is then released and eventually degraded
Group II Intron
Use an adenosine residue within the intron to initiate
splicing.
2’ OH of a specific adensosine in the intron acts as nucleophile attacking 5’ splice site to form lariat structure
A branched lariat
structure is formed as an
intermediate, and then
released.
Most introns require help to be spliced or removed by what large protein complex
spliceosome
Spliceosome contains five RNA-protein complexes involving small nuclear RNA proteins (snRNPs). What are they?
U1, U2, U4, U5, U6,
What is required to bring the spliceosome
together, but it is not needed for the
cleavage reaction?
ATP
What splicing generates alternative mRNAs from a single gene?
Alternative Splicing (intron could be spliced out by itself or spliced out along with an intervening exon)
Encoded as one or two long genes that are transcribed by RNA Pol I and then get spliced into the individual
rRNA molecules.
rRNA molecules also get methylated.
Some prokaryotes have tRNAs located in the sequences between
the rRNA genes.
Ribosomal RNAs
RNase P
a ribozyme
RNase D
a protein
Nucleotides are removed from both the 5’ and 3’-
ends of the initial transcript: the 5’ end is cut by RNase P (a ribozyme) and the 3’ end is cut by RNase D (a protein).
Base modification
5’ cleavage
3’ cleavage
“CCA” tail is added.
Additional splicing and base modifications occur
Transfer RNAs (tRNA)
Cellular level where cell can regulate the expression of proteins
mRNA is one way
TRUE/FALSE:
Bacterial mRNAs have an average half life of only 1.5
minutes.
TRUE
mRNA is degarded by what?
Ribonucleases
TRUE/FALSE:
In eukaryote, mRNA degradation usually starts with shortening of Poly A tail, then decapping
TRUE
TRUE/FALSE:
Average half life of a vertebrate mRNA is 3 hours, but it
can be degraded in seconds or days.
TRUE
