Exam 3 Flashcards
U2 snRNA
Bind to the branch point of the splicesome
Differences found in RNA but not DNA
Ribose sugar (less stable), uracil (additional pairing and unusual bases), and is single stranded (can form different shapes)
Base pairs in RNA (in order of decreasing strength)
G-C, A-U, and G-U
Base pairs in RNA (in order of decreasing strength)
G-C, A-U, and G-U
The function of uncommon bases in RNA
Usually found in non-coding tRNA (usually pseudouridine) and they are believed to stabilize RNA structure. They can modify base pairing if found in mRNA.
Name the non-watson-crick base pairings
C-G-C base triplet, T-A-A base triplet, T-A-T base triplet, and C-G-G base triplet
Coding RNAs and Non-coding RNAS: which feature is most important?
The sequence is most important for coding RNAs and the structure is most important for non-coding RNAs.
How are secondary RNA structures formed? What are their names?
By canonical base-pairing (non-watson and crick). Hairpin loop (@ the end), internal loop, stem, bulge, and external base (just one base sticks out)
Secondary structure vs tertiary structures
Typically easier to predict, more stable, and more evolutionarily conserved than tertiary structures.
Function of different RNAses
RNAse V1- cleaves dsRNA
RNAse 1 - cleaves ssRNA, 3’ end with a C or U
RNAse T1 - ssRNA, 3’ end with a G
Use of RNAse digestion assay
Can’t be used in live cells, but useful for 1 by 1 secondary structure interpretation. Very slow method for large RNA amounts.
Use of DMS seq
Assay for RNA secondary structure which can assess all cellular RNAs at once in-vivo (live cells). It modifies the As and Cs in ssRNA with DMS to show what the secondary structure looks like upon cDNA sequencing
Use of SHAPE-seq
Modifies the 2’-OH group of all ssRNA bases. Like DMS seq, it can be performed in vivo or in vitro and is genome-wide. Unlike DMS seq, you can identify the base pairing in hairpin structures and signal resolution is increased based on the burying of the base within the folded structure.
Tertiary structures of RNA
A form helix is a right-handed 11 bp/turn
B form helix is a right-handed 10 bp/turn
Duplex, triplex, G-quadruplex, pseudoknot, and kissing loops
What percentage of RNA is translated into protein?
<5%
Name 6 types of RNA based on their size/function/expression level
RNA genomes (ex: RNA viruses), protein-coding RNAs (mRNA), non-coding RNAs for translation (tRNA, rRNA), non-coding RNAs for RNA processing (gRNAs, snRNAs), non-coding RNAs for DNA replication (telomerase RNA), non-coding RNA for protein targeting (7SL RNA, 4.5S RNA)
Signal Recognition Particle (SRP)
Protein sorting RNA which directs membrane destined proteins for nuclear/rough er localization.
The SRP in eukaryotes is comprised of 7SL RNA
pRNA
Packing RNA is the most powerful molecular motor that packages RNA into viral capsids.
Ribozymes
RNAs that act as enzymes (natural or artificial). The sequences are able to form multiple secondary structures which can allow them to act in different ways.
Riboswitch
RNAs which can sense environmental changes and their functions can change as a result. Ex: bacterial RNA thermometer- only when the environment is above 42 degrees can the ribosome can bind to the RNA to create protein
Aptamer
RNA molecules that bind specific ligands and are usually less active than ribozymes. Selected through SELEX process.
What is the case for RNA being the first molecule to create life?
RNA is the only macromolecule that can store genetic information AND catalyze reactions. There were also once self-replicating RNAs that are no longer found in nature.
What are the alternatives that were proposed instead of RNA backbones in the first living organisms?
Treose (TNA), peptide (PNA), Glycerol (GDNA), and Pyranosyl RNA
RNA Transcription
Synthesis is 5’-3’, not restricted to S-phase, more error prone than DNA synthesis.
Operons
Very efficient bacterial DNA regions of related genes which can be turned on or off concertedly. multiple proteins with 1 RNA molecule
Bacterial RNA polymerase
Alpha, beta, beta’, and sigma are the main catalytic subunits. Sigma serves as a promoter recognition and is necessary for circular DNA there are several sigma-factors. Alpha causes chain initiation. Beta leads to chain initiation and elongation. Beta’ causes DNA binding. and
RNA polymerization: Initiation
1) The sigma-factor recognizes and binds the promoter to form a closed complex. 2) The RNA polymerase then unwinds the DNA to form an open complex transcription bubble (no helicase needed). 3) The first phosphodiester bond is formed between rNTPs to form an unstable ternary complex. 4) The sigma-factor is then released after ~10 nt to form a stable ternary complex.
Sigma promoter sequences
Usually a -35 sequence and -10 sequence (Pribnow box/TATA box). The strongest promoter sequence match the consensus. Some genes also have Upstream Promoters (UP) or enhancers that increase polymerase affinity.
DNA footprinting
Identifies RNA polymerase binding sites on DNA.
What does ChIP-seq do?
DNA binding proteins are introduced to a sample which bind to target DNA sequences. The DNA is then chopped into fragments. Antibodies for the proteins are then immunoprecipitated and isolated. DNA is released from the proteins and then is sequenced to identify the regions which bind to the proteins. Useful for identifying DNA modifications such as methylation/acetylation.
Abortive initiation
The first few rounds of polymerization are non-productive because sigma blocks the RNA exit channel. Several short strands of RNA are formed until sigma dissociates to form a stable ternary complex and productive transcription starts.
Termination sequence of bacterial transcription
GC rich inverted repeats cause the formation of a hairpin loop. The sequence then is followed by poly-A, which causes weak binding and then dissociation.
Rho-dependent termination of transcription
Rho is an ATP-dependent RNA helicase that unwinds the DNA:RNA hybrids. This is required for sequences without the terminator sequence (GC + A). Not all RNAs will terminate at the same base with RHO, causing different-sized transcripts.
Post-transcriptional modifications in bacteria
An addition of a poly-A tail causes RNA degradation (opposite of eukaryotes) because RNAses like the ssRNA. The hairpin 2 structure at the ends prevents degradation, but still incredibly unstable (which makes cotranslation a benefit)
Use of (q)RT-PCR
Common assay of RNA abundance on a limited number of RNAs (very low throughput, must be 1 RNA at a time). cDNA formation through PCR and then quantitatively monitor DNA amplification with intercalating dyes. Lower Ct value = higher abundance bc it crosses threshold faster.
Use of RNA-seq
Assay for RELATIVE abundance of RNA in the entire genome. From this method you can compare the cDNA sequence to the DNA sequence to identify the number of exons/introns.
RNA polymerase I
Nuclear eukaryotic RNA polymerase which transcribes rRNA only (80% of total RNA)
RNA polymerase II
Nuclear eukaryotic RNA polymerase which mostly transcribes mRNA (only 1-5% of total RNA). Structurally homologous to bacterial polymerase
RNA polymerase III
Nuclear eukaryotic RNA polymerase which transcribes tRNA and some small RNA sequences.
RNA Pol II (eukaryotic)
TFIID - Recognizes the TATA box and other sequences near the transcription starting point. ~12 subunits to regulate the start of transcription
TFIIH - unwinds DNA at the transcription start point. Phosphorylates Ser5 at the RNA polymerase CTD and releases RNA polymerase from the promoter.
Eukaryotic promoters
Difficult to predict due to wide variability. There is typically a core promoter ~4bp from TSS, proximal promoter ~200-40 bp from TSS, and an enhancer that is many kbs away from the TSS.
Eukaryotic enhancer
Activators bind to this sequence which is kb away from the TSS. A mediator is a large multiprotein complex that brings activators and basal transcription factors together to increase transcription. Insulators establish chromatin boundaries to help maintain the specificity of interactions despite the great distance between promoters.
Eukaryotic transcription initiation by poll II
TFIID (through Tata binding protein) binds the TATA box. TFIIB is then recruited to the TBP-TATA box complex to recruit RNA polymerase to the promoter. TFIIH serves as the helicase and phosphorylates the CTD to stimulate the release of the mediator. This causes the beginning of transcription.
Eukaryotic RNA elongation
Elongation factors help. The 7- methylG cap is added on the 5’ end to prevent degradation as it emerges. Splice junctions are marked by the binding of splice factors and then splicing occurs co-transcriptionally.
What does GRO-seq do?
Assay for active transcription by RNA pol. UTP is replaced with BrUTP which has specific antibodies for it. This can help identify alternative splicing mechanisms and led to the discovery that transcription can go in both directions.
Co-transcriptional modification of eukaryotic mRNA
Multiple phosphorylations of the CTD: unphosphorylated form is for activation, serine 5P causes early termination, & serine 2P causes late torpedo termination through polyadenylation.
Nucleosomes in eukaryotic rna transcription
Chaperone proteins can displace the octamer behind the polymerase, the polymerase can traverse without displacement, or the cotranscriptional histone modification can cause displacement.
Allosteric termination in eukaryotic RNA transcription
The binding of 3’ processing factors lead to rearrangement of the elongation complex and then termination
Torpedo termination in eukaryotic RNA transcription
A nuclease degrades the nascent RNA from the 5’ end, catching up with the elongation complex and then finally displaces poll II from DNA.
CTD/CTD cycle
The CTD of rpb1 contains many heptapeptide repeats: YSPTSPS. The CTD cycle is the patterned phosphorylation of the series at different stages of transcription.
Splicesome
5 subunit which removes the majority of introns. The major spliceosome cuts at 5’ GU and AG 3’. The minor spliceosome cuts at 5’ AU and AC 3’. THE BRANCH POINT IS ALWAYS AN A, forming a lariat structure. Cut by U6 RNA ribozyme and exon junction complexes mark the spliced sites.
Group II self-splicing
The 2’ OH group of an adenine attacks the 3’ splice site and forms a lariat. No spliceosome mechanism.
Group I self-splicing
The 3’ OH group of a guanine residue attacks the 5’ splice site and does not form a lariat.
tRNA splicing
An endonuclease cleaves the 3’ splice site and cleaves again at the ACC 5’ site. The ACC is retained and ligated. Maturation of tRNA is caused by the RNAse P.
T or F: Post-transcriptional base editings can happen in eukaryotes
T, it is most common in tRNA.
A-to-I editing
Commonly used in the synaptic transmission of neurons as a normal part of embryonic neuronal development.
C-to-U editing
ApoB in the liver normally is 4563 AA long. When C is changed to U via oxidative deamination in the intestine, the ApoB is 2153 AA long.
mRNA methylation in eukaryotes
Transient modification, most common is the m6A methylation. This usually is used to alter stability, translation, and modulate splice activity. Readers recognize & bind to the modified base, writers modify the base, erasers reverse the modification.
How are mature eukaryotic mRNAs exported from the nucleus?
Via the nuclear pore.
nonsense-mediated mRNA decay
Premature stop codons typically arise due to preserved introns. Exon junction complexes stay at the splicing sites on introns which is recognized as abnormal and targeted for degradation.
Non-stop decay
processes mRNA transcripts without a stop codon. These are typically caused by abortive transcription where the polymerase is removed before the full sequence is transcribed. The mRNA is stuck to the ribosome until proteins remove it and then endonucleases degrade it at the 3’ end.
No-go decay
The ribosome stalls due to strong secondary structure formation by intron retentions. Endonucleases cut the RNA in the middle and both parts get degraded since both ends don’t have the post-transcript capping.
Housekeeping RNA
rRNA, tRNA, snRNA, snoRNA, RNAse P, and 7SL RNA. Non-coding RNAs which are essential for cell survival
miRNA
type of ncRNA which is part of the RISC (RNA-induced Silencing Complex). It binds target mRNAs at the 3’ UTR and suppresses translation. Single active/leader strand imperfectly base pairs with the structure.
siRNA
miRNAs which signal the degradation of mRNAs. Perfect pairing with the target mRNA through the RISC pathway.
lncRNA
long-non-coding RNAs that are typically found within the introns or can form from bidirectional promoters of coding RNAs. Most have unknown functions but those characterized are implicated primarily in gene expression/chromatin structure. Often have very long half-lives and stay close to sites of transcription.
3 types of gene expression regulation in bacteria
Constitutive expression (always on), repressible (normally on, turned off by a repressor), and inducible (normally off, can be turned on by an inducer)
Lac Operon
Inducible operon in bacteria. Lac I creates repressor which bins to the operator. In the absence of lactose, the repressor protein is made and binds to the operator. This prevents polymerase binding. In the presence of lactose, the repressor binds to allolactose which allows polymerase binding.
Functions of the Lac genes
Lac I creates repressor which bins to the operator. Lac Z codes for b-galactosidase to form allolactose from lactose. Lac Y codes for the permease which brings lactose into the cell.
CAP binding
Necessary for the efficient transcription of lac genes. CAP requires cAMP to bind the binding site, which increases when glucose is low.
Trp operon
Repressible operon with two levels of control: repression and attenuation. Codes for the enzymes necessary to synthesize trp.
Trp operon: Repression
When Trp is low, the repressor can not bind and transcription proceeds. When Trp is high, the repressor forms a complex with the Trp and it can bind the operator to stop transcription.
Trp operon: Attenuation
When Trp mRNA is low, ribosome stalls and it favors the alternative loop so that transcription proceeds. When Trp mRNA is high, the sequence transcribes quickly and forms a terminator loop so transcription stops.
Chromatin and eukaryotic gene expression
Heterochromatin, nucleosome positions, and histone methylation can decrease transcription
Transcriptional co-regulators
Co-transcribed sequences on eukaryotic genes that can either activate or repress transcription by binding cooperatively to DNA.
Retinoid X Receptor (RXR)
3 closely related genes with multiple isoforms due to alternative splicing. A heterodimeric partner for other nuclear receptors to regulate cell differentiation, lipid and glucose metabolism, development, and immune response. Recruits co-regulators to activate or repress transcription.
