ch 8 (lectures 15-16) Flashcards
What is transcription, and what is the relationship between RNA and DNA in cells?
**RNA (Ribonucleic Acid) is made from a DNA template.
Transcription = the process of making RNA from DNA.
Transcript = the RNA produced.
What are the 4 ribonucleotides in RNA?
Purines: Adenosine-5’-monophosphate & Guanosine-5’-monophosphate
Pyrimidines: Cytidine-5’-monophosphate & Uridine-5’-monophosphate
What are the key differences between RNA and DNA?
Strandedness: RNA is single-stranded, while DNA is double-stranded.
RNA can fold into secondary structures, affecting its function.
Sugar Difference: RNA has an OH group at the 2’ position of ribose, making it less stable than DNA (which has H instead).
Base Difference: RNA uses Uracil (U) instead of Thymine (T).
- U pairs with A during transcription, but can **also pair with A or G in folded RNA.
Catalytic Ability: RNA can catalyze reactions; such RNA is called a ribozyme.
What are the two main classes of RNA and their functions?
mRNA (Messenger RNA)
- Encodes proteins.
- Generated in a process called translation.
Functional RNA
- Does NOT encode proteins.
- Involved in regulating gene function
How does RNA relate to the DNA template and nontemplate strands?
RNA is complementary to the template strand of DNA.
RNA is identical to the nontemplate (coding) strand, except U replaces T.
The nontemplate strand is also called the coding strand.
What is the +1 site, UTRs, and the role of the promoter in gene structure?
+1 site: Where transcription starts, located between the promoter and the first codon (usually ATG).
Promoter: A DNA region where RNA polymerase holoenzyme binds to initiate transcription.
* Some promoters are inactive in certain cells, leading to cellular differences despite identical DNA.
* Promoters are typically 50-100 bp from the +1 site, but not farther than 1 kb.
Untranslated Regions (UTRs):
* 5’ UTR: Before the AUG start codon.
* 3’ UTR: After the stop codon.
What are the key features of the prokaryotic promoter?
In prokaryotes, a typical promoter contains -35 and -10 consensus sequences.
The RNA polymerase sigma factor binds to these consensus sequences to initiate transcription.
What are the components and functions of the prokaryotic RNA polymerase holoenzyme?
β: Contains the catalytic site for RNA synthesis.
β’: Assists in catalysis.
σ (Sigma Factor): Binds to -10 and -35 consensus sequences.
* Helps in DNA unwinding.
* Called the specificity factor, as different σ factors target different promoters.
α & ω: Involved in regulation and assembly of the holoenzyme.
What is the transcription bubble in elongation, and where is it located?
The transcription bubble is the single-stranded DNA (ssDNA) region formed during transcription.
It is located inside the RNA polymerase.
What are the two main mechanisms of transcription termination in prokaryotes?
- Intrinsic Termination:
* Involves a stem-loop (hairpin) structure in the RNA, rich in G-C pairs, located upstream of a poly-U sequence.
* This structure, along with RNA polymerase (RNA pol) activity, leads to termination. - Rho-Dependent Termination:
* Involves a C-rich pausing signal in the RNA.
* A protein complex containing Rho binds to the RNA.
* The rut sequence upstream of the termination site is recognized by Rho to initiate termination.
What are the key differences between prokaryotic and eukaryotic transcription?
-
RNA Polymerases:
* Prokaryotes: One RNA polymerase.
* Eukaryotes: Three RNA polymerases. -
Transcription Factors:
* Prokaryotes: Sigma factors for initiation.
* Eukaryotes: Multi-subunit general transcription factors. -
Translation & Transcription:
* Prokaryotes: Co-transcriptional translation (transcription and translation occur simultaneously).
* Eukaryotes: Spatially separated transcription and translation (transcription in the nucleus, translation in the cytoplasm). -
Transcripts:
* Prokaryotes: Simple transcript (mRNA is ready to be translated).
* Eukaryotes: Processed transcript (includes cap, introns/exons, and poly-A tail). -
DNA Packaging:
* Prokaryotes: DNA is almost naked (not packaged in chromatin).
* Eukaryotes: DNA is packaged in chromatin (more complex packaging).
Where is RNA made in eukaryotic cells, and what evidence supports RNA moving from the nucleus to the cytoplasm?
RNA is made in the nucleus of eukaryotic cells.
- Evidence: RNA can move from the nucleus to the cytoplasm in eukaryotic cells, which is supported by observations of RNA being exported after transcription (visualized with pulses of radioactive, then chased with non-radioactive uracil)
What are the challenges in eukaryotic transcription?
Harder to Locate the Promoter:
* The genome is bigger, and genes are more spaced out.
* For example, one gene per 1.4 Kb in E. coli, one per 9 Kb in Drosophila, and one per 100 Kb in humans.
Transcription and Translation are Decoupled:
* Transcription occurs in the nucleus (nucleolus), while translation occurs in the cytoplasm.
DNA Packaging:
* Eukaryotic DNA is wrapped around histones, which must be removed or repositioned for transcription to occur.
Eukaryotic Transcription is More Complex:
* The process is more regulated and requires additional factors compared to prokaryotic transcription.
What are the three different RNA polymerases in eukaryotes, and what do they transcribe?
RNA Polymerase II:
* Transcribes mRNA and some functional RNA.
RNA Polymerase I:
* Transcribes all rRNA except 5S rRNA.
* Only makes functional RNA (does not encode proteins).
RNA Polymerase III:
* Transcribes tRNA, 5S rRNA, and snRNA.
* Only makes functional RNA (does not encode proteins).
How do the three eukaryotic RNA polymerases recognize promoters and initiate transcription?
Each RNA polymerase (I, II, III) has unique promoters and general transcription factors (GTFs).
* GTFs (General Transcription Factors) are protein complexes that help RNA polymerases recognize promoters and initiate transcription.
How does RNA Polymerase II initiate transcription in eukaryotes?
RNA Polymerase II transcribes mRNA and some functional RNA.
General Transcription Factors (GTFs) assist Pol II in recognizing and initiating transcription at the promoter.
* TFIID is a key GTF that binds to the TATA box
* TATA-less promoters use elements like INR (initiator) elements to direct Pol II.
Steps of Transcription Initiation:
* TFIID binds the promoter at the TATA box via TBP.
* Other transcription factors (TFs) associate, forming the preinitiation complex.
* RNA Polymerase II begins RNA synthesis and leaves the promoter, leaving TFIID behind.
* This process is coupled to the phosphorylation of RNA Pol II’s C-terminal domain (CTD).
What are the key transcriptional processing steps of RNA in eukaryotes?
5’ Capping:
* 7-methylguanosine is attached to the 5’ end with three phosphate groups.
* Function: Protects RNA from degradation and is required for translation.
Polyadenylation (Poly-A Tail):
* Polyadenylation sequence (AAUAAA/AUUAAA) is recognized.
* Adds a Poly-A tail (AAAAAA…) to the 3’ end.
* Function: Protects RNA from degradation and stimulates translation.
Splicing:
* Removes introns, keeping only exons in the mature mRNA.
🔹 These processes were traditionally called post-transcriptional, but they actually occur co-transcriptionally for most genes.
What is the function of the 7-methylguanosine (m7G) cap in eukaryotic mRNA?
Functions of the 7-methylguanosine (m7G) cap:
* Protects RNA from degradation by exonucleases.
* Acts as a binding site for proteins that assist in translation.
What is polyadenylation, and what are its functions in eukaryotic mRNA?
Polyadenylation:
* Addition of 50–250 adenosine (A) nucleotides to the 3’ end of mRNA.
* Recognized by the highly conserved hexanucleotide sequence (AAUAAA).
Functions:
* Protects mRNA from decay by exonucleases.
* Promotes translation by interacting with the translation machinery.
What is the difference between an Open Reading Frame (ORF) and a Coding Sequence (CDS)?
Open Reading Frame (ORF):
* The nucleotide sequence from start codon (ATG) to a stop codon (TAA/TGA/TAG).
* Does NOT include the stop codon itself.
Coding Sequence (CDS):
* The portion of the ORF that appears in the final mRNA (T replaced with U in RNA).
In Prokaryotes: CDS = ORF (no introns).
In Eukaryotes: ORF includes introns, while CDS is the processed exonic sequence.
* CDS+introns=ORF
How does alternative splicing contribute to protein diversity in eukaryotes?
Alternative Splicing allows for greater protein diversity from a single gene by rearranging the exons during mRNA processing.
* 70% of human genes undergo alternative splicing.
* More complex organisms (like humans) tend to have more introns per gene, which increases the potential for diverse splicing outcomes.
* In humans, more than 70,000 types of proteins can be produced from just 21,000 genes.
Pre-mRNA (Nascent RNA):
Refers to RNA that has not yet been processed, including splicing, 5’ capping, and polyadenylation.
What are the conserved sequences involved in intron splicing and the role of snRNPs?
Conserved Sequences for Intron Splicing:
* GU at the 5’ end of the intron.
* AG at the 3’ end of the intron.
* A is located closer to the 3’ end of the intron.
* These motifs are highly conserved within species.
snRNPs (Small Nuclear Ribonucleoprotein Complexes):
* snRNPs are involved in the recognition of specific RNA sequences during splicing.
* snRNPs include U1, U2, U3, etc., and they play a crucial role in catalyzing the removal of introns.
How does the spliceosome assemble and function in RNA splicing?
- U-snRNPs bind in a stepwise manner to the pre-mRNA.
- The RNA component of the U-snRNPs:
Base pairs with mRNA and other U RNA molecules.
Brings the necessary residues for catalysis into the correct position and timing. - Some RNAs in the spliceosome are directly involved in the catalysis of intron removal.
- Some U-snRNPs leave after the catalysis.
- Formation of a lariat structure (looping of the intron).
- Lariat is released, and the exons are joined together to form the mature mRNA.
Chemical Reactions in Splicing:
* 2’ OH group of the branch point A attacks the 5’ phosphate of the intron, breaking the bond between the 5’ end of the intron and creating a 5’ to 2’ bond, forming the lariat.
* 3’ OH group of the 5’ phosphate attacks the 3’ end of the intron, creating a 5’ to 3’ bond (splicing event), and the lariat is released.
What are the different types of functional RNA and their roles?
tRNA (Transfer RNA):
* Carries amino acids to the ribosome for protein synthesis.
* Transcribed by RNA polymerase III in eukaryotes.
rRNA (Ribosomal RNA):
* RNA components of the ribosome.
* Have structural and enzymatic activities.
* Transcribed by RNA polymerase I, except for 5S rRNA which is transcribed by pol III.
snRNA (Small Nuclear RNA):
* Involved in splicing of eukaryotic RNAs into mRNA.
* Transcribed by pol II and III.
miRNA (Micro RNA):
* Processed from larger RNAs.
* Regulates translation and RNA stability, playing a key role in gene regulation and infection control.
siRNA (Small Interfering RNA):
* Part of the RNA interference (RNAi) pathway.
* Helps prevent the mobilization of transposons and viruses.
piRNA:
* Small RNAs that help control transposition (movement of transposons).
