106 Questions
What is the primary function of the Dicer enzyme in RNA interference?
To cleave larger dsRNAs into smaller fragments
What is the main difference between the effects of miRNA and siRNA?
miRNA prevents translation of mRNA, while siRNA degrades mRNA
What is the purpose of the RNA-induced silencing complex (RISC)?
To unwind double-stranded RNA and select the antisense strand
What is the role of the Argonaut protein in RNA interference?
To guide the RISC complex to the targeted mRNA and facilitate binding
What is the characteristic of the pre-miRNA that is transcribed directly from a gene?
The presence of a hairpin loop
Why is RNA silencing believed to be an evolutionarily conserved process?
Because it is a mechanism to fight off foreign invaders such as viruses and transposons
What is the outcome when siRNA is a perfect match for the mRNA target?
The mRNA is degraded by the Argonaut protein
What is the result of creating strains of mice that lacked the proteins that make miRNA?
The mice developed a variety of health problems, including heart disease and cancer
What is the purpose of the ATP-dependent process in RISC?
To unwind double-stranded RNA and select the antisense strand
What is the main function of RNA interference?
To defend against foreign invaders such as viruses and transposons
What is the estimated percentage of the transcriptional output of our genomes that is comprised of non-coding RNAs (ncRNA)?
98%
What is the primary function of micro RNAs (miRNA) and small interfering RNA (siRNA)?
Control of gene expression
What is the source of small interfering RNAs (siRNA)?
From exogenous sources, such as a viral infection, a transposable element, or a molecule supplied by a scientist
What is the length of micro RNAs (miRNA)?
22 nucleotides
What is the enzyme responsible for cutting micro RNAs (miRNA) from a larger RNA?
Dicer
What is the significance of the discovery of let-7 miRNA in many species, including humans?
It is a fundamental process of gene regulation
What is the role of non-coding RNAs (ncRNA) in gene regulation?
They regulate many processes, including gene silencing, replication, RNA processing, and more
What is the significance of the transcription of non-coding RNAs (ncRNA) in higher organisms?
It is responsible for the complexity of higher organisms
What is the role of miRNA-101 in prostate cancer?
It leads to the overexpression of a particular histone methyltransferase
What is the implication of micro RNAs in heart conditions?
They influence the production of alpha and beta myosin heavy chain
What is RNA activation (RNAa)?
A process of activating a gene
What type of interactions do proteins that bind to DNA use during transcription?
Hydrogen bonding, electrostatic attractions, and hydrophobic interactions
What is the function of the DNA-binding domain in proteins?
To bind to DNA during transcription
What is the most common motif found in DNA-binding proteins?
Helix-turn-helix (HTH)
What is the role of the major groove in DNA-binding proteins?
It is the site of binding for proteins that recognize DNA with specific base sequences
What is the role of glycine in the HTH motif?
It is involved in the beta-turn
What is the role of glutamine and arginine in DNA-binding proteins?
They interact favorably with adenine and guanine
What is the effect of A-T rich areas on the structure of DNA?
It causes local variations in helix structure
What is the purpose of the zinc-finger domain in DNA-binding proteins?
To recognize the shape of the bases protruding into the grooves
What is the characteristic of the basic-region leucine zipper motif?
The motif has a series of leucines every seven residues
What is the function of the acidic domains in transcription factors?
To recognize other transcription factors
What is the purpose of the glutamine-rich domains in transcription factors?
To recognize other transcription factors
What is the primary function of the polyadenylate tail at the 3' end of a message?
To protect the mRNA from nucleases and phosphatases
What is the function of the proline-rich domain in the activator CTF-1?
To bind to histone proteins via the proline repeats
What is the term for the DNA sequences that are expressed in the final mRNA product?
Exons
What is the characteristic of the zinc-finger domain in DNA-binding proteins?
The motif has two closely spaced cysteines and two closely spaced histidines
What is the function of the basic region in the basic-region leucine zipper motif?
To bind to DNA
What is the result of the splicing reaction in eukaryotic genes?
The removal of introns and the linking of exons
How many introns are present in the pro a-2 collagen gene in chickens?
51
What is the purpose of the transcription-activation domains?
To recognize other transcription factors
What is the characteristic of the protein TFIIIA?
It has nine repeating structures of 30 amino acids each
What is the term for the process of removing intervening sequences from a primary transcript?
Splicing
What is the function of the C-terminal end of the activator CTF-1?
To bind to histone proteins
What is the function of the 5' cap in mRNA?
To protect the mRNA from exonuclease degradation
What is the characteristic of the genes of eukaryotes?
They are not necessarily continuous
What is the purpose of the poly-T tail in affinity chromatography?
To isolate mRNA from a cell lysate
What is the consequence of inaccurate splicing in eukaryotic genes?
The formation of abnormal proteins
What is the term for the ribonucleoprotein particles that form through association with a set of nuclear proteins?
RNPs
What is the conserved sequence at the branch site within the intron in higher eukaryotes?
PyNPyPuAPy
What is the result of the nucleophilic attack on the phosphodiester backbone at the 5' splice site?
Formation of a lariat structure and release of exon 1
What is the function of small nuclear ribonucleoproteins (snRNPs) in the splicing process?
To mediate the splicing process
What is the characteristic of the RNA portion of snRNPs in higher eukaryotes?
It is between 100 and 200 nucleotides in length
What is the function of the spliceosome in the splicing process?
To mediate the splicing process
What is the estimated percentage of proteins in humans that have isoforms based on alternative splicing?
5% of proteins
What is the primary function of the motifs described in the transcription factors section?
To determine the role of a new protein as a transcription factor
What is the role of regulatory proteins in alternative splicing?
To direct the alternative splicing
What is the result of differential splicing of the human troponin T gene?
Sixty-four possible isoforms of the protein
What is the purpose of the leader and trailer sequences in RNA transcripts?
To facilitate the trimming process
What is the enzyme responsible for generating the 5' ends of all E. coli tRNAs?
RNase P
What is the characteristic of the Tau protein in people with Alzheimer's disease?
It has six isoforms
What type of modification takes place in eukaryotic tRNA and is not found in prokaryotes?
Addition of a 2'-O-methylribosyl group
What is the mechanism by which the splice sites are brought together in both self-splicing and ribonucleoprotein-mediated splicing?
A lariat mechanism
What is the purpose of the CCA sequence at the 3' end of tRNA molecules?
To provide a binding site for amino acids
What is the primary difference between the processing of rRNA in prokaryotes and eukaryotes?
The degree of trimming
What is the purpose of capping the 5' end of eukaryotic mRNA?
To increase the stability of the mRNA
What is the purpose of polyadenylating the 3' end of eukaryotic mRNA?
To increase the stability of the mRNA
Why is mRNA modified after initial transcription in eukaryotes?
To increase the stability of the mRNA
What is the primary difference between the processing of mRNA in prokaryotes and eukaryotes?
The presence of a poly-A sequence
What is a characteristic of Group I ribozymes?
They require an external guanosine, which becomes covalently bonded to the splice site
What is the significance of the discovery of RNAs that catalyze their own self-splicing?
It provides evidence for the existence of an RNA-based world
What is a characteristic of hammerhead ribozymes?
They can be catalytically active with as few as 43 nucleotides
What is the role of the folded conformation of RNA in ribozymes?
It is crucial for the catalytic activity of the ribozyme
What is the significance of the discovery of RNA that catalyzes its own self-splicing in relation to the splicing of mRNA by snRNPs?
It is easy to see a connection between the two processes
What is the role of divalent cations in the catalytic activity of ribozymes?
They stabilize the folded structure of the RNA by neutralizing some of the negative charges on the phosphate groups
What is a characteristic of the transesterification reaction that takes place in self-splicing RNA?
It releases one end of the intron
What is the significance of the discovery of RNA that catalyzes its own self-splicing in relation to the origin of life?
It suggests that RNA may have been the original genetic molecule and the original catalytic molecule
What is the characteristic of the catalytic efficiency of catalytic RNAs compared to protein enzymes?
It is lower than that of protein enzymes
What is the requirement for the catalytic activity of Group II ribozymes?
The 2'-OH of an internal adenosine
What is the primary function of RNA polymerase in transcription?
To catalyze the process of nucleotide incorporation
What is the role of the s-subunit in RNA polymerase?
To recognize promoters
What is the direction of RNA synthesis during transcription?
5' to 3' on the coding strand
What is the function of the Pribnow box in prokaryotic transcription?
To recognize promoters
What is the role of enhancers in transcriptional regulation?
To stimulate transcription above basal levels
What is the difference between a promoter and an enhancer?
A promoter is a DNA sequence near the transcription start site, while an enhancer is a DNA sequence farther away from the start site
How does repression work in the lac operon?
The repressor binds to the operator and blocks RNA polymerase
What is the role of RNA secondary structures in transcription attenuation?
To regulate transcription through secondary structures
What is the difference between the template strand and the coding strand?
The template strand is the strand that is transcribed, while the coding strand is the strand that is not transcribed
What is the function of Mg2+ in RNA synthesis?
To facilitate the binding of RNA polymerase to the template strand
What is the role of chromatin remodeling complexes in eukaryotic transcription?
To allow RNA polymerase to access the promoter region and begin transcription
What is the function of the TATA box in eukaryotic promoters?
To aid in promoter recognition and binding
What is the role of general transcription factors in transcription initiation?
To aid in promoter recognition and binding
What is the function of response elements in transcription?
To increase the rate of transcription above basal levels
What is the difference between micro RNA and small interfering RNA?
Micro RNA is produced by transcription of the cell’s genes, while small interfering RNA is from exogenous sources
What is the role of RNA-induced silencing complex (RISC) in RNA interference?
To bind to the mRNA with the target sequence
What is the function of DNA-binding domains in transcription factors?
To bind to the DNA and aid in promoter recognition
What is the role of histone modifying enzymes in eukaryotic transcription?
To allow RNA polymerase to access the promoter region and begin transcription
What is the outcome when the sequence of the siRNA is perfect?
The mRNA is degraded before it gets a chance to be translated
What is the role of transcription-activation domains in transcription factors?
To bind to other proteins and increase the rate of transcription
What is the primary function of the 5' cap in mRNA?
To protect the mRNA from degradation
What is the term for the process of removing intervening sequences from a primary transcript?
Splicing
What is the characteristic of a ribozyme?
It is a RNA molecule with catalytic ability
What is the role of the branch point in the splicing reaction?
It is the site where the lariat shape is formed
What is the function of the 3' polyadenylation in mRNA?
To stabilize the mRNA
What is the characteristic of the genes of eukaryotes?
They are non-continuous
What is the role of snRNP in the splicing reaction?
It is the ribonucleoprotein molecule that facilitates the reaction
What is the consequence of inaccurate splicing in eukaryotic genes?
The mRNA is degraded
Study Notes
Non-Coding RNAs (ncRNAs)
- 98% of transcribed RNA does not make protein
- ncRNAs linked to processes like transcription, gene silencing, replication, RNA processing, modification, translation, protein stabilization, and translocation
Micro RNAs (miRNAs) and Small Interfering RNAs (siRNAs)
- small double-stranded RNAs involved in controlling gene expression
- miRNAs:
- Produced by transcription of cell's genes
- About 22 nucleotides long
- Cut from larger RNA by enzyme Dicer
- Not a perfect match for mRNA target, preventing translation
- siRNAs:
- From exogenous sources (viral infection, transposable element, or supplied by scientist)
- Cut by Dicer to 21-25 nucleotides
- Guides RISC complex to targeted mRNA, facilitating binding and silencing
RNA Interference (RNAi)
- Process by which miRNA and siRNA decrease gene expression
- RISC complex mediates RNAi
- Extent of sequence matching controls effect on gene expression
Where miRNA Fits into Gene Expression
- Similar process to RNA interference, using same enzymes
- miRNA loaded onto RISC complex, interacting with mRNA target
- No cleavage of mRNA, preventing translation
Evolutionary Significance of RNA Silencing
- Protects genome from foreign invaders (viruses, transposons)
- Conserved process in higher organisms
- Importance demonstrated in mouse studies (health problems without miRNA) and human studies (miRNA and cancer)
DNA-Binding Proteins
- Bind to DNA during transcription using interactions like hydrogen bonding, electrostatic attractions, and hydrophobic interactions
- Two functional domains: DNA-binding domain and transcription-activation domain
Structural Motifs in DNA-Binding Proteins
- Helix-turn-helix (HTH), zinc fingers, and basic-region leucine zipper (bZIP) motifs
- Interact with DNA in major or minor groove
- HTH motif: a-helix fits into major groove
- Zinc fingers: looped out from intersection of cysteines and histidines with zinc ion
- Basic-region leucine zipper: basic region binds to DNA in major groove, leucine zipper interacts with second analogous protein fragment
Transcription-Activation Domains
- Involved in binding of transcription factors to other proteins, not DNA
- Motifs like acidic domains, glutamine-rich domains, and proline-rich domains
Posttranscriptional RNA Modification
- Modification of RNA after transcription
- Trimming, addition of terminal sequences, and base modification occur
- tRNA, rRNA, and mRNA undergo modification
- tRNA:
- Trimming of precursor sequence
- Addition of terminal sequences (C CA at 3' end)
- Base modification (methylation, sulfur substitution)
- mRNA:
- Capping of 5' end with guanylate residue
- Polyadenylating 3' end
- Splicing of coding sequences (in eukaryotes)### Intron and Exon Structure
- Intron: a sequence of DNA that is removed from the primary RNA transcript to produce mature RNA
- Exon: a sequence of DNA that is retained in the mature RNA
- The pro-alpha2 collagen gene in chickens has 51 exons, with the coding regions spread out over 40,000 base pairs
Splicing Mechanisms
- Splicing occurs in the nucleus and involves the formation of ribonucleoprotein particles (RNPs) with a set of nuclear proteins
- The substrate for splicing is the capped, polyadenylated pre-mRNA
- Splicing requires cleavage at the 5' and 3' ends of introns and the joining of the two ends
- Specific sequences make up the splice sites, with GU at the 5' end and AG at the 3' end of introns in higher eukaryotes
- A branch site within the intron also has a conserved sequence, with PyNPyPuAPy (where Py is any pyrimidine and Pu is any purine) and an invariant A
Alternative RNA Splicing
- Many proteins are always spliced in the same way, but some can be spliced in different ways to give different isoforms of the protein
- In humans, 5% of proteins produced have isoforms based on alternative splicing
- Alternative splicing is regulated by specific proteins that affect the recognition of splice sites
- The human troponin T gene produces a muscle protein with many isoforms due to differential splicing
Ribozymes
- Ribozymes are RNA molecules with catalytic activity
- The discovery of ribozymes has had a profound impact on the way biochemists think about catalysis
- Ribozymes can catalyze their own self-splicing, as well as other reactions involved in protein synthesis
- The catalytic efficiency of ribozymes is less than that of protein enzymes
- Ribozymes have been found to stimulate transcription elongation
Characteristics of Ribozymes
- Several groups of ribozymes are known to exist, including Group I and Group II ribozymes
- Group I ribozymes require an external guanosine and use a transesterification mechanism
- Group II ribozymes display a lariat mechanism of operation, similar to the mechanism seen in splicing facilitated by snRNPs
- Hammerhead ribozymes are the smallest known ribozymes, with as few as 43 nucleotides
Basics of Transcription
- RNA synthesis is the transcription of the base sequence of DNA to that of RNA
- All RNAs are synthesized on a DNA template
- The enzyme that catalyzes the process is DNA-dependent RNA polymerase
- The RNA chain grows from the 5' to the 3' end
- The enzyme uses one strand of the DNA (the antisense strand, or template strand) as the template for RNA synthesis
RNA Polymerase Subunits
- In prokaryotes, the subunits of RNA polymerase (α, β, β', and ω) make up the core of the enzyme and are responsible for the enzymatic activity
- The σ-subunit is used for promoter recognition
- In eukaryotes, the opposite strand is often used to produce small noncoding RNAs
Transcription Control
- Frequency of transcription is controlled by the promoter sequence
- Additional sequences upstream can also be involved in regulating transcription
- Proteins called transcription factors can bind to these sequences and stimulate or inhibit transcription
- Many prokaryotic genes are controlled in groups called operons, and expression of some genes is controlled by transcription attenuation
Repression and Induction
- Repression involves the binding of a repressor protein to the operator portion of the promoter, preventing transcription
- Induction involves the binding of an inducer molecule to the repressor, allowing transcription to occur
RNA Secondary Structures
- In prokaryotes, transcription and translation are linked
- RNA secondary structures can form during transcription, affecting the speed of translation
- Depending on the speed of translation, the RNA produced can form different hairpin loop structures that can act as terminators or allow transcription to proceed
Test your knowledge of RNA interference, a process of gene silencing that involves the degradation of specific mRNA molecules. This quiz covers the functions of Dicer, miRNA, siRNA, RISC, and Argonaut proteins.
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