Ch. 21 Flashcards
What are the 4 bases in RNA?
- Adenine
- Uracil
- Cytosine
- Guanine
Draw adenine.
Draw guanine.
Draw cytosine.
Draw thymine.
Draw uracil.
Draw the modified nucleotides.
Compare and contrast DNA and RNA.
DNA
- Thymine
- Deoxyribose sugar
RNA
- Uracil
- Ribose sugar
- Complex intrastrand structures
- Can form ribozymes (catalytic molecules)
Why is RNA a highly dynamic molecule?
- RNA undergoes a cycle of synthesis, functional interaction & degradation
- Its structure is altered by binding of ligands
- Base pairing between mRNA and ncRNA can modulate protein synthesis process
What do the 3 types of RNA needed for protein synthesis do (mRNA, rRNA, tRNA)?
- mRNA: carrier of genetic info
- rRNA: major constituent of ribosomes
- tRNA: adaptor molecule that connects RNA synthesis to protein synthesis
How are transcription and translation different in prokaryotes vs eukaryotes?
Prokaryotes
- Both transcription and translation occur in the same compartment –> mRNA is bound by ribosomes as soon as it’s transcribed
- Transcription and translation are tightly coupled
Eukaryotes
- Transcription occurs in nucleus, translation occurs in cytoplasm
- Requires additional factors to transport RNA from one compartment to the next
What does monocistronic mean?
Encodes a single protein
What does polycistronic mean?
Encodes multiple proteins on a single mRNA
Is eukaryotic mRNA monocistronic or polycistronic?
Most often monocistronic
- Coding sequence is often discontinuous due to introns (don’t code for protein) interrupting exons (protein-coding)
Explain eukaryotic mRNA processing.
- Introns must be removed from precursor mRNA via RNA splicing to generate an ORF
- 5’ terminus of mature mRNA m7G cap
- 3’ terminus contains poly A tail
- m7G cap and the poly A tail facilitate interaction between mRNA and ribosome, increasing translational efficiency
What kinds of organisms have proportionally higher ncRNA?
Multicellular organisms (but inversely related to proportion of protein-coding genes)
What are the 5 way lncRNA are located in the genome?
What are the 4 modes of action lncRNA has in mediating cellular functions?
- Base pairing between nucleotides in the lncRNA and the target RNA
- Base pairing between lncRNA and single-stranded regions of DNA
- Formation of functional ribonucleoprotein complexes similar to ribosomes and spliceosomes
- Ligand-induced riboswitches that function in signaling pathways
Compare and contrast the structure of prokaryotic and eukaryotic RNA polymerases.
- Both have the same 5 core subunits
- Prok: also has α2ββ′ω structure
- Euk: another 7 auxiliary protein subunits
What is the cAMP receptor protein (CRP)?
Bacterial transcription regulatory protein that upon binding cAMP binds to specific DNA sequences and stimulates transcription
What are σ factors?
- Bind to DNA sequences known as -35 and -10 boxes
- Binds bacterial RNA polymerase and is required for the initiation of transcription
How do σ factors work?
Targets RNA polymerase to the promoter by decreasing the affinity of the protein for nonspecific DNA sequences by a factor of 104
What are housekeeping genes?
Encode proteins and RNA involved in essential biochemical processes common to most all cells
What is DNase I footprinting?
In vitro lab method for identifying DNA sequences that can function as binding site for sequence-specific DNA binding proteins
Why are the -35 and -10 boxes called that?
Protected sequences were located ~10 and ~35 nucleotides upstream of the transcription start site
What is the TATA binding protein (TBP)?
Binds to the sequence 5′-TATAAAA-3′ located ∼30 nucleotides upstream of the RNA polymerase II transcription start site
What is the structure of the TATA binding protein?
- Single polypeptide chain that folds to form 2 symmetric halves
- Halves flank DNA and generate a sharp bend as a result of 2 Phe residues on each side that insert into the minor groove and force a kink into the DNA backbone
What does RNA synthesis use to generate an RNA trascript?
DNA template strand (DNA must be unwound by RNA polymerase in order to access the template strand)
What are the steps of transcription?
- RNA polymerase, directed by its bound σ factor, binds to the promoter
- A closed complex and an open complex form in succession - Transcription is initiated within the complex, leading to a conformational change that converts the complex to the elongation form followed by movement of the transcription complex away from the promoter (promoter clearance)
- The σ subunit dissociates stochastically (at random) as the polymerase enters the elongation phase
What is a closed complex?
Inactive form of bacterial RNA polymerase holoenzyme (bound DNA is intact)
What is an open complex?
Active form of bacterial RNA polymerase holoenzyme (bound DNA is intact but partially unwound near -10 sequence)
What are the 3 main RNA processing steps from primary transcript to mRNA?
- 5’ capping: by guanine-N7 methyltransferase
- Splicing
- Polyadenylation (in the end, the exons are joined to form a continuous sequence that specifies a functional polypeptide)
What is the function of 5’ capping?
- Protection against ribonucleases (less rapid degradation) i.e. prolongs mRNA lifetime
- Aids in export from the nucleus and in translation
What is splicing?
Multiple proteins derived from a single gene
- Exons retained/skipped
- Introns excised/retained
- Exon-intron splice sites altered
- Alterations in transcription start site or poly-A addition
What are the effects of alternate splicing?
- Soluble vs membrane-bound proteins
- ± phosphorylation sites
- Subcellular targeting information
- ± allosteric effector site
- Affinity of a ligand for a receptor
Explain polyadenylation.
- Most mRNAs have a poly-A tail that is 80-250 nucleotides long
- Serves as a binding site for specific proteins
- Confers a distinctive 3’ end structure
- Poly-A tail & associated proteins likely help protect the mRNA from enzymatic degradation
- Poly-A tail is added in a multistep process; mediates transcription termination
- Transcript is extended beyond the site where the poly-A tail is to be added
- Then it is cleaved at the poly-A addition site by an endonuclease at highly specific sites: 5’-AAUAAA-3’ which is about 30 nucleotides upstream of the cleavage site
When are ribozymes regenerated?
After the trans cleavage of target RNA
What do ribozymes do?
Mediate intramolecular or intermolecular cleavage reactions of RNA substrates
What are the 2 primary mechanisms proposed for ribozyme-mediated phosphodiester cleavage?
- Acid-base catalysis
- Two-metal ion catalysis
What is the hammerhead ribozyme?
Catalytic RNA that is capable of both cis and trans cleavage
How are intronic or intergenic sequences from eukaryotic mRNA, rRNA, or tRNA removed?
By an RNA-catalyzed reaction using either:
- A self-splicing cis reaction
- A trans-splicing reaction mediated by spliceosomes
What are spliceosomes?
Ribonucleoprotein complex that excises introns from mRNA and splices together the exons to form a mature mRNA
What are group I introns?
- Class of catalytic RNAs that use a guanosine cofactor to self-splice the introns and join the exons to form mature RNA
- Found in precursors to rRNA, tRNA, and mRNA
- Not in vertebrates
What are group II introns?
Class of catalytic RNAs that don’t need a guanosine cofactor for self-splicing, and instead use a cis-acting adenine residue within the intron
What are 2 notable differences between group I and group 2 introns?
- Group II introns do not require an exogenous nucleoside to initiate the cleavage reaction
- Group II excised intron forms a lariat structure in which the 5′ end of the intron is linked to the 2′-OH of an adenosine residue near the 3′ end of the intron
Diagram the mechanism of spliceosome-mediated trans splicing of precursor mRNA transcripts.
What do introns removed by spliceosomes from primary mRNA transcripts contain?
Short conserved sequences at the 5′ (GU) and 3′ (AG) splice sites, as well as in the branch site
What are small nuclear ribonucleoproteins (snRNPs)?
A complex of small nuclear RNAs and associated proteins that compose a spliceosome
Diagram and explain the spliceosome assembly pathway.
- Capped pre-snRNA transcripts are exported from the nucleus to the cytoplasm
- Each snRNA is assembled with a heptameric ring of proteins, called Sm proteins, to form the snRNP core complex
- snRNA is hypermethylated on the 5’ cap and transported back to the nucleus
- snRNP is assembled into a spliceosome complex
What are common modifications in tRNA?
What is small nucleolar RNA (snoRNA)?
Small RNA molecules that form part of the ribonucleoprotein complexes in the nucleolus that aid in processing rRNA molecules
What are the 2 classes of alternative splicing mutations?
- Those that disrupt the use of alternative splice sites (cis effects)
- Those that affect expression of spliceosome components or regulatory proteins (trans effects)
What is the most frequent effect of alternative splicing mutations?
Exon skipping and occurs as a result of a base change at one of the conserved regions that must be recognized by the spliceosome complex
What is retinitis pigmentosa?
Disease of the retina resulting in progressive retinal degeneration, which in some cases is caused by mutations in genes that encode spliceosome components
Diagram mRNA decay.
