Chapter 10: From DNA to Proteins: Transcription and RNA Processing Flashcards
What creates instability in RNA, which is not present in DNA?
The OH-group on the 2’ carbon
What allows RNA molecules to form secondary structures?
Hydrogen bonding between complimentary bases on the same strand
Which classes of RNA carries the genetic code for proteins? What do the other classes do?
- ONLY mRNA
- Other classes solely function at the RNA level
What types of RNA are expressed in bacteria and eukaryotes?
- rRNA
- mRNA
- tRNA
What is the function of rRNA?
- Structural and functional components of the ribosome
- Used during the translation process
What is the function of mRNA?
- Carries genetic code for protein
- Transcribed from DNA
What is the function of tRNA?
Helps incorporate amino acids into the polypeptide chain during protein synthesis
What are small RNA molecules mostly involved in? Which cell type are they found in?
- Mostly involved in the processing or the regulation of RNA molecules
- Allows them to control the expression of certain genes
- Solely Eukaryotes
What does CRISPR function with? What is it synthesized by?
- CRISPR-RNA (small RNA molecule)
- Only synthesized by Prokaryotes
What is transcription?
The synthesis of an RNA molecule from a DNA template
What kind of structures do DNA molecules undergoing transcription exhibit?
- Christmas tree-like structures
- Multiple components function on a single gene to produce various RNA molecules (branches)
- Multiple RNA polymerases may act on the same gene
Compare the different classes of RNA molecules in Prokaryotes and Eukaryotes.
- RNA molecules found in Prokaryotes and Eukaryotes: mRNA, rRNA, tRNA
- RNA molecules only found in Eukaryotes: pre-mRNA, snRNA, miRNA, siRNA
Do both DNA strands undergo transcription at the same time?
- No, RNA is transcribed from one DNA strand (template strand)
- The non-template strand is not usually transcribed
What is the direction of the synthesis of RNA?
- 5’ –> 3’
- New nucleotides are added to the 3’-OH group
Where does the template strand occur: bottom or top strand?
- The template strand may occur from either
- Top (-) or bottom (+)
- Not all genes possess templates on the same strand
How does transcribed RNA compare to the template strand of DNA?
RNA synthesis is complimentary and antiparallel to the template strand
Define a gene.
Transcription unit that includes a promoter, an RNA-coding region, and a terminator
Where is the RNA-coding region located?
Between the transcription start site and the termination site
What is the function of the transcription termination site?
Halts the actions of the enzymes responsible for transcription
What is the function of the transcription start site?
Allows the polymerase to attach to the DNA and commence transcription
Define a promoter region.
- DNA sequence that promotes and regulates transcription
- Possesses multiple motifs (5-6 nucleotides), which are recognized by transcription factors
Where is the promoter region found in relation to the RNA-coding region?
- Promoter region: upstream
- RNA-coding region: downstream
How does the synthesized RNA compare to the non-template strand?
- Resembles the non-template strand
- However, uracil is present in RNA instead of thymine in DNA
What are the substrates used in RNA synthesis?
Ribonucleoside triphosphates
Where are nucleotides added during transcription?
3’ end
What catalyzes the addition of primers in transcription?
- NOTHING
- Unlike DNA replication, there is not need for primers in transcription
Which enzyme is responsible for the addition of RTPs during transcription?
RNA polymerase
What happens to the transcription bubble as transcription occurs?
- The bubble does not expand
- It simply moves alongside the DNA strand
What questions does the promoter region regulate?
When, where, and how many transcripts are needed
What subunits does the core bacterial RNA polymerase consist of?
- Two beta subunits
- Two alpha subunits
- One omega subunit
What must the core bacterial RNA polymerase interact with to become an RNA polymerase holoenzyme?
The Sigma factor
Is RNA polymerase sufficient for transcription?
- No, it creates the space between two strands of DNA
- A basal rate (minimum amount of transcript produced) may occur, but multiple proteins are necessary for proper transcription
What is a consensus sequence?
Consists of the most commonly encountered bases at each position in a group of related sequences
What are RNA polymerases and transcription factors made to detect?
- Detects signature motifs
- When they encounter these consensus sequences, they bind to DNA
Pyrimidines are indicated by what?
Y
Purines are indicated by what?
R
Where are consensus sequences found in bacterial promoters?
- Upstream of the start site
- Approximately at positions -10 and -35
What does the holoenzyme bind to in the bacterial promoter?
The -35 and -10 consensus sequences
What is released as the RNA polymerase moves beyond the bacterial promoter?
Sigma factor
How is bacterial RNA transcription initiated?
When core RNA polymerase binds to the promoter with the help of the sigma factor
What are the two major ways to terminate transcription in Prokaryotes?
- Hairpin structure
- Rho protein
How does termination of transcription in Prokaryotes occur with the hairpin structure?
- Inverted repeat regions at the end of the gene are complimentary to each other
- Stimulates the formation of a hairpin (secondary structure), which stalls the RNA polymerase
- Hydrogen bonds between the RNA molecule and DNA template break, allowing the RNA transcript to separate from the template
How does termination of transcription in Prokaryotes occur with the rho protein?
- Rho binds to an unstructured region of RNA and moves towards the 3’ end
- When RNA polymerase encounters a terminator sequence, it pauses and rho catches up
- Upon helicase activity, rho unwinds the DNA-RNA hybrid and brings transcription to an end
How many RNA polymerases are there in Eukaryotes?
Five
Which RNA polymerases are present in all eukaryotes, and which are only present in plants?
- All Eukaryotes: RNA polymerase I, II, III
- Plants: IV and V
Which RNA polymerase is the most solicited? Why?
- RNA polymerase II
- Responsible for the transcription of the genes encoding for proteins (pre-mRNA)
What is the function of RNA polymerase I?
Ribosomal RNA synthesis
What is the function of RNA polymerase III?
- tRNAs
- Small RNA molecules
What is the function of RNA polymerase IV?
Some siRNAs
What is the function of RNA polymerase V?
RNA molecules taking part in heterochromatin formation
The promoters of genes transcribed by RNA polymerase II in Eukaryotes consist of what regions?
- Core promoter
- Regulatory promoter containing consensus sequences
What are two difference between the promoter region in Eukaryotes and Prokaryotes?
- The promoter region extends BEYOND the transcription start site in Eukaryotes
- There are promoter regions that are located hundreds or thousands of base pairs away
Where are the core promoters in Eukaryotes located?
Within the first 50 nucleotides from the RNA coding region
What is the TATA box?
- In Eukaryotes, it is the attachment site for the RNA polymerase
- The TATA box activates a basal amount of transcript made, but the conserved motifs (elements) may increase the transcription
What is the general model for transcription in Eukaryotes?
- There is no general model for transcription, as each gene is different in Eukaryotes
- Different contexts may also influence the way genes are regulated
What is Rat1?
Endonuclease
What is the function of Rat1?
Requirement for termination of transcription by RNA polymerase II in Eukaryotes
How does termination of transcription in Eukaryotes occur with Rat1?
- RNA polymerase II transcribes well past the coding region of most genes
- Cleavage is near the 3’ end of the RNA, while RNA polymerase continues transcribing
- Rat1 attaches to the 5’ end of the trailing RNA and moves towards the RNA polymerase, degrading RNA as it goes
- When Rat1 reaches the polymerase, transcription is terminated
How many genes do humans possess?
Between 24 000 and 30 000 genes
How many proteins do humans express?
60 000 proteins
How can 30 000 genes generate 60 000 proteins?
Certain genes may code for multiple proteins due to the concept of non-collinearity
How are RNA molecules read?
Codons (3 nucleotides)
What does the co-linearity principle state?
That a continuous sequence of nucleotides in the DNA codes for a continuous sequence of amino acids in the protein
Are Prokaryotic genes collinear or non-collinear? What about Eukaryotic genes?
- Prokaryotic: collinear
- Eukaryotic: non-collinear
How was the non-collinearity of Eukaryotic genes discovered?
- By hybridizing DNA and mRNA
- When DNA binds to RNA, non-coding regions are seen in a loop
- Whatever is in the loop is NOT found in RNA
- Thus, coding sequences must be interrupted by non-coding regions
What are the coding sequences of Eukaryotic genes disrupted by?
Non-coding introns
Differentiate introns and exons.
- Introns: non-coding regions
- Exons: coding regions
What is RNA splicing? What is its function?
- The removal of introns
- Splicing may be used to create multiple protein variants
Are introns present in Prokaryotes?
No
What are the three primary regions of the mature mRNA?
- 5’ untranslated region
- Protein-coding region
- 3’ untranslated region
- These regions must NOT be spliced out to create a functional protein
What are the three kinds of RNA processing methods in Eukaryotes?
- Splicing
- 5’ cap
- 3’ poly(A) tail
What is 5’ capping? What bond catalyzes the capping?
- Phosphate groups on the 5’ end of the mRNA are capped with the nucleotide “7-methylguanine”
- Attached to the pre-mRNA by a unique 5’-5’ bond
What is the function of 5’ capping?
- RNA becomes more stable with the cap (less prone to degradation in the cytoplasm)
- Aids in the translation processing
What is the poly(A) tail added solely to?
Messenger RNA, allowing for their identification
What is the function of the poly(A) tail?
- Recognized as mRNA in the translation process
- Help protect the transcript from degradation
What is the 3’ poly(A) tail?
Addition of multiple adenosine molecules through cleavage and polyadenylation to the 3’ end of mRNA
Where is the Shingo-Dalgarno sequence found? What is it required for?
- Found solely in Prokaryotes
- Required to commence protein translation
Where does RNA processing occur? When?
- In the nucleus
- Prior to their transportation to ribosomes for translation
What are the three primary functions of RNA processing?
- Increases stability of the RNA
- Guides the translation process
- Diversifies the number of proteins that may be generated from an RNA molecule
Is the poly(A) tail present in the DNA sequence of a gene?
No, it is added following transcription
What are the coding sequences of Eukaryotic genes disrupted by? Are they present in Prokaryotes?
- Non-coding introns
- They are NOT present in Prokaryotes
What does splicing of pre-mRNA require?
Consensus sequences
Where are the critical consensus sequences for splicing of pre-mRNA?
- 5’ splice site
- 3’ splice site
Where are the weak consensus sequences for splicing of pre-mRNA?
At the adenine branch point
What do most introns start and end with?
- Start: GU (5’ end)
- End: AG (3’ end)
Where does RNA splicing take place?
Within the spliceosome
What is the mechanism of splicing?
- G of the GU pairs with the A at the branch point, forming a lariat
- Lariat is cut at the 3’ splice site, and the intron is released as a lariat
- Two exons are spliced together
What is alternative processing controlled by?
Spliceosome
How may alternative splicing produce two different proteins?
- Splicing can remove introns
- Alternative splicing can remove introns and an exon, creating a similar smaller protein
What occurs in multiple 3’ cleavage sites?
- The organism may select to cut at either cleavage site, depending on the environmental context
- mRNA products are different lengths, creating diversity from a single gene
Where is tRNA found? What is its major function?
- Found in the cytoplasm
- Incorporate amino acids into polypeptide chains (translation)
What is the common secondary structure of all tRNAs? What is it maintained by?
- Cloverleaf structure (three arms)
- Maintained by hydrogen bonds
How many bases are comprised in an anticodon? What does it interact with?
- Three bases
- Interacts with a codon
How do ribosomes differ in Prokaryotes and Eukaryotes?
- Prokaryotes: smaller (70S)
- Eukaryotes: larger (80S)
How does the number of rRNA genes differ between organisms?
- Differ widely
- E. coli: 1
- Human: 280
What organisms are miRNA and siRNA found in?
Eukaryotes
What organisms is CRISPR RNA found in?
Bacteria
What did David Baulcombe discover?
- Very small RNA molecules in a plant
- Found out they controlled immunity (viral infections)
What are siRNAs and miRNAs produced from?
Double-stranded RNAs
Describe the mechanism for the production of RNA-inducing silence complex (RISC).
1) Transcription through an inverted repeat in the DNA produces an RNA molecule that folds to produce dsRNA
2) dsRNA is cleaved by the Dicer enzyme to produce miRNA
3) One strand of miRNA combines with proteins to form a RISC
What does RISC become in the miRNA’s case? What is the effect?
Combines with proteins and inhibits translation
What does RISC become in the siRNA’s case? What is the effect?
Combines with proteins and degrades the mRNA
Does a gene encode miRNA or siRNA?
Gene encodes miRNA
How does dsRNA arise in siRNA if there are no genes encoding for them?
From RNA viruses or long hairpins
What are the overall effects of miRNA and siRNA?
- Cause silencing of the transcript
- Involved in regulating gene expression
Which class of RNA is correctly paired with its function?
A) Small nuclear RNA (snRNA): processes rRNA
B) Transfer RNA (tRNA): attaches to an amino acid
C) MicroRNA (miRNA): carries information for the amino acid sequence of a protein
D) Ribosomal RNA (rRNA): carries out RNA interference
B) Transfer RNA (tRNA): attaches to an amino acid
Explain the components of the Christmas-tree-like structures of DNA undergoing transcription.
- Trunk: portion of the DNA molecule
- Branches: RNA molecules that have been transcribed from DNA
What is the difference between the template strand and the non-template strand?
- Template strand: DNA strand that is transcribed into an RNA molecule
- Non-template strand: not transcribed
The transcription apparatus moves (upstream/downstream) as transcription takes place.
downstream
What is the function of the sigma factor in bacteria?
- Controls the binding of RNA polymerase to the promoter
- Without sigma, RNA polymerase will initiate transcription at a random point along the DNA
How many types of RNA polymerases do bacteria and eukarya possess?
- Bacteria: single type
- Eukarya: several distinct types
What steps does initiation comprise?
1) Promoter recognition
2) Formation of a transcription bubble
3) Creation of the first bonds between rNTPs
4) Escape of the transcription apparatus from the promoter
What does the orientation and spacing of consensus sequences on a DNA strand determine?
Which strand will be the template for transcription, and thereby determine the direction of transcription
An RNA molecule possesses, at least initially, ______ phosphate groups at its 5’ end.
three
What are two common features do Rho-independent terminators?
- They contain inverted repeats, which bind to each other during transcription, forming a hairpin
- Their transcription produces a string of uracil nucleotides after the hairpin in the transcribed RNA
What is the consequence of the string of uracil nucleotides in Rho-independent terminators?
- Causes the RNA polymerase to pause, allowing time for the hairpin structure to form
- The hairpin destabilizes the DNA-RNA pairing, causing transcription to terminate
Transcription is a selective process. What does this mean?
Only parts of DNA are transcribed at any one time
What evidence indicated that eukaryotic genes are not colinear with their proteins?
When DNA was hybridized to the mRNA transcribed from it, regions of DNA did not correspond to RNA looped out
Are introns transcribed in RNA?
Yes, but they are later removed by RNA processing (splicing)
What is the function of the Shine-Dalgarno sequence in bacterial mRNA?
Serves as the ribosome-binding site during translation
What is the consequence of deleting or mutating the adenine nucleotide branch point?
Prevents splicing
If a splice site were mutated so that splicing did not take place, what would the effect be on mRNA?
A) It would be shorter than normal
B) It would be longer than normal
C) It would be the same length but would encode a different protein
B) It would be longer than normal
When are siRNAs and miRNAs produced?
When larger double-stranded RNA molecules are cleaved by the enzyme Dicer
Which of the following phrases does not describe a function of the promoter?
A) Serves as sequence to which transcription apparatus binds
B) Determines the first nucleotide that is transcribed into RNA
C) Determines which DNA strand is template
D) Signals where transcription ends
D) Signals where transcription ends
What binds to the −10 consensus sequence found in most bacterial promoters? A) The holoenzyme (core enzyme + sigma) B) The sigma factor alone C) The core enzyme alone D) mRNA
A) The holoenzyme (core enzyme + sigma)
What is the difference between the core promoter and the regulatory promoter?
A) Only the core promoter has consensus sequences.
B) The regulatory promoter is farther upstream of the gene.
C) Transcription factors bind to the core promoter; transcriptional activator proteins bind to the regulatory promoter.
D) Both b and c
D) Both b and c
How are the processes of RNA polymerase II termination and rho-dependent termination in bacteria similar and how are they different?
- Both processes use a protein that binds to the RNA molecule and moves down the RNA toward the RNA polymerase
- They differ in that rho does not degrade the RNA, whereas Rat1 does so
What forms the transcription bubble?
- NOT gyrase and helicase
- RNA polymerase itself
Why are pre-mRNAs capped, but tRNAs and rRNAs aren’t?
A protein that adds the 5′ cap is associated with RNA polymerase II, which transcribes pre-mRNAs but is absent from RNA polymerase I and III, which transcribe rRNA and tRNAs.
Alternative 3′ cleavage sites result in A) multiple genes of different lengths. B) multiple pre-mRNAs of different lengths. C) multiple mRNAs of different lengths. D) all of the above.
C) multiple mRNAs of different lengths.
