Lecture 3: RNA (from dna to protein) Flashcards
Explain the central dogma of molecular biology
DNA —(transcription)—-> RNA—–(translation)—> protein
What is the chemical structure of RNA/ what makes RNA different from DNA?
The sugar in RNA is a ribose (not a deoxyribose), so it actually has a hydroxyl on the 2’ carbon
Uracil in RNA instead of Thymine
There are still phosphodiester bonds between nucleotides to create a backbone (like DNA)
RNA is single stranded but can fold into 3D structures
Draw the differences between ribose and deoxyribose
Draw the difference between uracil and thymine
RNA has the ability to ________.
RNA has the ability to fold into itself
Explain the function of the following RNA types:
- mRNA
- rRNA
- tRNA
- snRNA
- snoRNA
- miRNA
- siRNA
- mRNA: codes for proteins
- ribosomal RNAs, form structure of ribosome
- transfer RNA, critical to protein synthesis as adaptors between mRNA and AA’s
- small nuclear RNAs, splice pre mRNA
- miRNAs regulate gene expression by blocking translation of selective mRNAS
- siRNAs turn off gene expression by directing degradation of selective mRNAs
Explain the role of RNA Polymerase.
Does it require a promotor?
What is its error rate?
RNA Polymerase: catalyzes the formation of phosphodiester bonds between ribonucleotides
No promotor is required (more errors)
1 in 10^4 is the error rate
Note: the RNA Poly actually reads the 3 prime to 5 prime end because the pre mRNA reflects the coding strand
There is also a short region of DNA/RNA helix within the RNA Poly
What are the signals encoded in DNA to initiate and terminate trascription?
What does RNA Poly I, II, and III do?
Promotor initiates transcription
Terminator (A-T forming a hairpin) stops transcription
RNA Poly I: transcribes rRNA genes
RNA Poly II: transcribes all protein coded genes
RNA Poly III: transcribes tRNA genes
Explain how the initiation of transcription starts with RNA Poly II and its transcription factors
TATA box: located 25 nucleotides upstream
- The TBP subunit of TFIID binds the TATA box, enabling TFIIB binding
- The rest of the tx factors, incliuding RNA poly II assemble at the promoter
- TFIIH then uses ATP to pry apart DNA at the double helix at the tx starting point
- TFIIH ALSO phosphorylates the C terminal domain of RNA Poly II, changing its conformation so that the polymerase can release from general factors and begin elongation phase
What are the roles of TFIID and TFIIH in transcription initiation?
TFIID: the TBP regonizes the tata box, and the TAF recognizes other DNA sequences near the starting point and regulates DNA binding by TBP
TFIIH: unwinds DNA at the transcription starting point, phosphorylates Ser 5 of the RNA Poly II CTD, which releases RNA Poly from the promoter
What do transcriptional activator proteins do?
Transcriptional activator proteins determine the rate and pattern of transcription. They act from very very far away (thousands of nucleotides away sometimes) and they help RNA Poly, the general transcription factors, and the mediator all to assemble at the promoter.
Explain the processing of pre mRNA to mature mRNA in Eukaryotes. What are the three things that are ESSENTIAL for the mature mRNA to get exported out of the cell nucleus?
- 5’ cap
- RNA splicing
- poly A tail
These steps are important. If any of them don’t happen, the mRNA won’t get exported out of nucleus and into cytoplasm and therefore won’t get expressed
Give a brief overview of RNA splicing mechanism.
There are several snRNAs involved, but basically U1 recognizes the 5’ splicing site, U2 recognizes the region to make a loop, the 3’ end is recognized. The intron forms a loop called a lariat, and then gets excised. The two exon regions are then united
How can humans make so many proteins from one gene?
RNA SPLICING
Also the fact that we can have three different reading frames plays a role as well
As a disease, Beta thalessemia is an issue with
RNA splicing
What is the start codon? What are the stop codons?
How many reading frames are there?
Start codon: AUG (methionine)
Stop Codons: UAA, UAG, UGA
Three possible reading frames
What do aminoacyl-tRNA synthases do? Do they have a proofreading mechanism?
tRNA synthases attach the AA to the tRNA using ATP hydrolysis.
Yes it does have a proofreading mechanism. They can remove incorrect AA’s
Explain the basic steps on how mRNA is translated into a protein in a ribosome.
So you have a large ribosomal subunit and a small ribsomal subunit. There are three sites, E, P, and A.
Steps:
- tRNA binding at the A site
- peptide bond formation between AA in A site and the peptide chain
- large subunit translocation
- small subunit translocation
What do elongation factors do?
Elongation factors bring tRNAs into proper position in the A site
How does translation get initiated?
How does translation stop?
Translation initiation:
start codon/Met is recognized by intiation factors
Then Met tRNA binds to the P site of the small ribosomal subunit
That then binds to the 5’ end of mRNA
Translation stop:
Stops at the stop codons UAA, UAG, UGA
Release factors bind to the A site
Polypeptide is released and then ribosome dissociates
How do antibiotics inhibit protein synthesis?
They block translation
What is the role of HsP70 proteins?
They aid in proper protein folding (need ATP to do so)
What are the quality control mechanisms?
- 5’ cap and poly A tail
- Exon junction complex, stimulates translation
- nonsense mediated mRNA decay
- protein folding and degradation
