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The set of steps that readies the newly-transcripted mRNA molecules for translation into proteins
RNA processing
Do prokaryotes only, eukaryotes only, or both go through RNA processing?
eukaryotes only
When does RNA processing occur to eukaryotic mRNA?
after transcription but before translation
What are the 3 main steps to RNA processing?
5’ cap addition, 3’ polyadenylation, splicing
To what type of eukaryotic RNA does RNA processing happen?
mRNA
The ___ steps of RNA processing happen ________________ to ____________ _______ of the _______ molecule during the _____________ phase (aka while ______________ is still going)
3, simultaneously, different parts, mRNA, elongation, transcription
RNA Processing Step #1: 5’ cap addition
The 5’ end of the ________ mRNA is CAPPED by ___-_______________. This step is catalyzed by the __________ _________ which has __ enzymatic activities and is in the same family as _____ __________.
nascent, 7-methylguanosine, Capping Enzyme, 3, DNA Ligase
RNA Processing Step #1: 5’ cap addition
3 Steps/Enzymatic Activities:
___________ removes 5’ ______-____________ from the NTP at the ___ end of the ___-_______ (aka the first NTP)
Specific enzymatic activity: _________
Release of: ___
___________ of ___-________ through __-__ triphosphate bridge → ____ released
Specific enzymatic activity: _______
Addition of: _____
Release of: _____
___________ of the __________ base at position ___ via _____ methyl donor
Specific enzymatic activity: _________
hydrolysis, gamma-phosphate, 5’, pre-mRNA, RTPase, Pi, guanylation, pre-mRNA, 5’-5’, PPi, GTase, GTP, PPi, methylation, guanosine, N7, SAM, N7MTase
RNA Processing Step #1: 5’ cap addition
Capping occurs during ____________. The capping enzyme is recruited by and binds to the ______ ___ ______________ ___-________ ________ (____)
elongation, RNAP II phosphorylated C-terminal domain, CTD
RNA Processing Step #1: 5’ cap addition
Occurs in _____________ only. ______________ have a normal 5’-PPP.
eukaryotes, prokaryotes
RNA Processing Step #1: 5’ cap addition
Why add the cap? (3)
protects the end from getting chewed up by ribonucleases, helps export it out of the nucleus, important for translation because ribosomes in eukaryotes recognize and bind to the cap
RNA Processing Step #1: 5’ cap addition
A common modification within the cap is that the _____ ___ NTPs of the ___-______ (aka at the ___ end), if the base is _________, may be _____________ at the ___ position of the base. This means the ____ functional group becomes an ________ functional group.
first 2, pre-mRNA, 5’, adenine, methylated, N6, OH, O(CH3)
RNA Processing Step #1: 5’ cap addition
What are the 3 overall steps/enzymatic activities?
hydrolysis, guanylation, methylation
Which step of RNA processing is this image associated with?
5’ cap addition
RNA Processing Step #2: 3’ polyadenylation
The 3’ end of the __________ mRNA is polyadenylated, aka a ______ ___ _______ is added. Essentially, a bunch of ___________ are added to the end of the mRNA strand. This step is catalyzed by _______ ___ ___________, which does not use a ___________ but does use _____.
nascent, poly (A) tail, adenines, Poly (A) Polymerase, template, ATP
RNA Processing Step #2: 3’ polyadenylation
This step mostly occurs in ______________.
eukaryotes
RNA Processing Step #2: 3’ polyadenylation
In eukaryotes, mRNA initially do not have ______ ____________ ________ like they do in bacteria. The RNA Polymerase II only ______ once it reaches a __________ _______, which is a point in the mRNA molecule where the ___________ actually ________.
strict termination signals, stops, cleavage signal, message, cleaves
RNA Processing Step #2: 3’ polyadenylation
What 6 nucleotides make up the cleavage signal?
AAUAAA
RNA Processing Step #2: 3’ polyadenylation
RNAP II has to go _____ the __________ _______ in order for it to be _____________. This means that the actual ____________ _____ is ~ ___ bases ____________ from the cleavage signal.
past, cleavage signal, recognized, termination site, 30, downstream
RNA Processing Step #3: Splicing
________ = the sections of the pre-mRNA that get removed
introns
RNA Processing Step #3: Splicing
________ = the sections of the pre-mRNA that remain to be incorporated into the mature mRNA molecule (aka the actual coding sequences)
exons
RNA Processing Step #3: Splicing
Which tend to be longer: introns or exons?
introns
RNA Processing Step #3: Splicing
This step is catalyzed by the _____________. It is one of the biggest macromolecules in the cell, with ~ ___ proteins. It is composed of _________ (______ __________ __________________). It has ~ ___ _________ molecules that range up to a couple ____________ nucleotides long, which is considered quite ______ for RNA. They fold into ____ __________ that usually involve some hairpin loop base-pairing within the snRNA as well as base-pairing to parts of the ___-_______ molecule. These _____ parts are what helps with catalysis by ____________ the _______ ___________ sequences and __________ to the sequences before and after to ensure ___________ for the ______________ attacks.
Spliceosome, 50, snRNPs, small nuclear ribonucleoproteins, 5 snRNA, hundred, small, 3D structures, pre-mRNA, RNA, recognizing, splice junction, binding, alignment, nucleophilic
What is this a picture of?
Which step in RNA processing is it involved in?
snRNP within the Spliceosome, splicing
__________ Gene = the gene that encodes the major protein in an egg that sustains the embryo as it’s developing
ovalbumin
To convert the Ovalbumin ______ into the ________ Ovalbumin _______, the ______ has to first go through ____________ and then _____________, with _________, ________________, and finally _________.
gene, mature, mRNA, gene, transcription, processing, capping, polyadenylation, splicing
RNA Processing Step #3: Splicing
An experiment was done on the __________ gene where we ___________ an mRNA molecule to the DNA from which it was transcribed to show how much of the ________ _____ _____________ was _____ and that these _____ sequences (aka ________) come from the ________ of the gene, not the _____.
Ovalbumin, hybridized, original DNA sequencing, lost, lost, introns, middle, end
RNA Processing Step #3: Splicing
Do introns come from the middle or the end of a gene?
middle
RNA Processing Step #3: Splicing
Where are the sequences for splicing encoded?
within the gene itself
RNA Processing Step #3: Splicing
___ nucleotides at the beginning and end of an ______ are _________ (i.e. always the ______).
___ & ___ are always at the START of an intron just downstream from the ___ splice site
___ & ___ are always at the END of an intron just upstream of the ___ splice site
2, intron, invariant, same, G, U, 5’, A, G, 3’
15% of genetic disease mutations are related to __________
splicing
____________ __________ = from a single gene, you can make a lot of slightly different versions of the same protein
alternative splicing
RNA Processing Step #3: Splicing
Some transcripts exhibit _______-_________ splicing. This is a form of ___________ splicing that is specific to a particular _______ and occurs more often in ________ eukaryotes.
tissue-specific, alternative, tissue, higher
What is one way that gives eukaryotes additional complexity beyond just the # of our genes?
alternative splicing
RNA Processing Step #3: Splicing
The Lariat Mechanism
___ ____ on a particular _________ within the intron attacks the phosphodiester bond at the ___ _______ ______. This results in the release of _____ ___ with a free ___ ____. The ______________ keeps it from diffusing away upon release.
___ ____ on the first _____ of Exon 1 attacks the phosphodiester bond at the ___ ________ ______. This results in the release of the ______ in lariat form and the re-formed mRNA with ______ ___ connected/spliced to ______ ___.
(see pic)
2’ OH, adenine, 5’ splice site, Exon 1, 3’ OH, Spliceosome, 3’ OH, GTP, 3’ splice site, intron, Exon 1, Exon 2
RNA Processing Step #3: Splicing
Which mechanism are most introns removed by?
lariat mechanism
RNA Processing Step #3: Splicing
Is the 5’ splice site located at the START or END of an intron?
start
RNA Processing Step #3: Splicing
Is the 3’ splice site located at the START or END of an intron?
end
RNA Processing Step #3: Splicing
The Lariat Mechanism
Does not use _____ but does use the ______________.
ATP, Spliceosome
RNA Processing Step #3: Splicing
The Lariat Mechanism
Which part of this mechanism is an example of chemistry that DNA can’t do?
nucleophilic attack by a 2’ OH
RNA Processing Step #3: Splicing
Self-Splicing Mechanism
_______ ___ introns are _______________ → they splice themselves!
They don’t need the _____________ or any proteins → the _____ is catalytic (recall the beginning-of-the-world hypothesis)
Group I, autocatalytic, spliceosome, RNA
RNA Processing Step #3: Splicing
Self-Splicing Mechanism
What type of eukaryotic RNA can sometimes do this?
ribosomal RNA (rRNA)
RNA Processing Step #3: Splicing
Self-Splicing Mechanism
What was this the first example of?
RNA catalysis
RNA Processing Step #3: Splicing
Self-Splicing Mechanism
3’ OH of _________ attacks the phosphodiester bond at the splice junction connecting the ____ ______ to the Intron. This results in the bond between the ____ ______ and the Intron being _______. However, the ______ folds in on itself, meaning it is able to _____-_____ with the ____ _______ to insure it does not diffuse away.
3’ OH of the ____ NTP on the ____ _____ attacks the phosphodiester bond at the splice junction connecting the _____ ______ to the Intron. This results in the bond between the _____ ______ and the Intron being ________ and removes the _____-_______ between the Intron and the ____ ______. The two ______ are now connected/spliced together.
The newly-released 3’ OH at the end of the intron attacks an inner portion of the intron, forming a ______ and releasing a little bit of the Intron, as well.
(see pic)
guanine, left exon, left exon, cleaved, intron, base-pair, left exon, last, left exon, right exon, right exon, cleaved, base-pairing, left exon, exons, circle
What are the most abundant macromolecules in the cell?
proteins
The vast majority of enzymes are _________
proteins
Up to ____% of a cell’s energy can be directed toward protein synthesis
90
___% of an E. coli cell’s dry mass can be ribosomes → almost _____ new ribosomes are made per E.coli cell per minute!
25, 1000
Many important antibiotics target _________ ____________
protein synthesis
The Genetic Code
The Big Question:
Protein Synthesis, aka _____________, uses a completely different kind of ____________ than what we’ve become familiar with. How do we switch from the ____________ used in DNA ______________ and RNA ______________ to the ____________ used in Translation of _________?
translation, chemistry, chemistry, replication, transcription, chemistry, proteins
the set of RULES by which a linear sequence of nucleotides specifies the linear sequence of a polypeptide
genetic code
the instructions contained in a gene that tell a cell how to make a specific protein
genetic code
The Genetic Code
It is made up of _______, which are three-letter chains of ______________. Each _______ codes for one specific _______ ______. The code determines the ______ in which _______ ______ are added to a _____________ chain during protein synthesis. Therefore, the genetic code dictates the _________ of _______ ______ in a protein.
codons, nucleotides, codon, amino acid, order, amino acids, polypeptide, sequence, amino acids
The Genetic Code
Amino acids with ________ _______ tend to be more common in proteins
multiple codons
The Genetic Code
The genetic code was not made at ________, but was selected through evolution. So the amino acids grouped together tend to have similar _________ ___________.
random, physical properties
The Genetic Code
Reading along a DNA or RNA strand in one direction, there are ___ possible reading frames, with only ___ making sense. In double-stranded DNA, there are ___ possible reading frames: ___ on the top strand and ___ on the bottom strand, again with only ___ usually making sense.
3, 1, 6, 3, 3, 1
The Genetic Code
A 2+ change in reading frame is equivalent to a ___ change in the reading frame.
-1
The Genetic Code
A -1 change in reading frame is equivalent to a ___ change in the reading frame.
2+
The Genetic Code
How did Crick figure this out?
He used __________, an _____________ agent, to make small _____ mutations, which he posited could potentially change the ________ ______.
He compared the ____________ of each individual _____ AND the _____________ that arose when they were ___________.
Through these experiments, Crick showed that the genetic code had to be in multiples of ___, that there was no “____________”, AND inferred the presence of ______ __________.
proflavin, intercalating, indel, reading frame, phenotypes, indel, phenotypes, combined, 3, punctuation, stop codons
The Genetic Code
Crick’s Frameshift Manipulations:
What happens to the reading frame after +1 nucleotide insertion?
-1 shift in the rest of the reading frame
The Genetic Code
Crick’s Frameshift Manipulations:
What happens to the reading frame after +1 nucleotide insertion & a deletion of 1 nucleotide?
reading frame is restored
The Genetic Code
Crick’s Frameshift Manipulations:
What happens to the reading frame after +1 nucleotide insertion & +2 nucleotide insertion?
reading frame is restored
The Genetic Code
What are the 3 main types of point mutations?
What is the overall classification of mutation that these usually fall into?
missense, nonsense, indel frameshifts, loss-of-function
The Genetic Code
Types of Point Mutations
___________ = when a single mutated nucleotide changes the codon into one that now codes for a different amino acid
missense
The Genetic Code
Types of Point Mutations
__________ = when a single mutated nucleotide changes the codon into a stop codon
nonsense
The Genetic Code
“___ ___ ___ _____________” = the A purine has changed to a G purine
A to G transition
The Genetic Code
“___ ___ ___ _____________” = the T pyrimidine has changed to an A purine
T to A transversion
Which of the following is NOT a form of RNA processing of eukaryotic RNA polymerase II primary transcripts?
A. polyadenylation
B. proteolytic cleavage
C. 5′-end capping
D. splicing
B
The poly(A) tail on eukaryotic mRNAs…
A. is located 20-30 nucleotides on the 5′ (upstream) side of the polyadenylation addition sequence (AAUAAA)
B. begins at the site where RNA polymerase II falls off
C. does not require a template for synthesis
D. is synthesized by RNA polymerase II
C
Which of these processes does NOT occur in the nucleus of eukaryotes?
A. pre-mRNA splicing
B. polyadenylation
C. translation
D. 5′-end capping of mRNAs
C
Which of the following is TRUE about spliceosomes that carry out the splicing of eukaryotic pre-mRNAs?
A. ATP is required for the cleavage and ligation reactions
B. The first cleavage step of splicing results in the formation of an intron lariat intermediate
C. The spliceosome acts in the cytoplasm
D. The spliceosome consists of five proteins and about 50 RNAs
B
Which of the following statements is NOT TRUE about the 5′-end cap structure of mature eukaryotic mRNAs?
A. The cap protects the mRNA from degradation.
B. The cap is a residue of 7-methyluridine.
C. The cap is methylated.
D. It participates in the binding of the mRNA to the ribosome to initiate translation.
B
The insertion of three nucleotides into an open reading frame cannot result in…
A. a nonsense mutation.
B. a missense mutation.
C. the destruction of a stop codon.
D. a frameshift mutation.
D
Which statement about mRNA splicing is FALSE?
A. The process is carried out by the spliceosome.
B. Splicing can generate different mRNAs from a single gene.
C. The lariat forms when a ribose 2’-OH within the intron attacks the phosphoester bond at the 5’ splice site, breaking the old phosphoester bond and making a new one.
D. The lariat RNA becomes part of the mature mRNA.
E. The 2 nucleotides at the 5’ and 3’ ends of the introns are nearly invariant.
D
Mutagen X causes G to A mutations on the sense strand, while mutagen Y causes A to T mutations on the sense strand. Which mutagen would you expect to have the most deleterious effect on the protein? (see slide 20)
A. X
B. Y
C. They would have the same effect
D. Not enough info to answer this
E. None of the above
B
The insertion of one nucleotide in the coding sequence of a gene is most likely to lead to which of the following mutations?
A. Frameshift mutation
B. Missense mutation
C. Nonsense mutation
D. Silent mutation
E. Transversion mutation
A
If Crick’s experiment HAD revealed that one + mutation could be suppressed by adding 3 more + mutations to the gene, what would this indicate about the genetic code?
A. The genetic code is multiples of 2
B. The genetic code is multiples of 3
C. The genetic code is multiples of 4
D. The genetic code is overlapping
E. None of the above
C
You synthesize an RNA molecule with U and C nucleotides used at a 3:1 ratio and then use this synthetic RNA for an in vitro translation reaction. What would be the amino acids in this protein in descending quantity from most to least? (math on slide 19)
A. Leu, Ser, Pro, Phe
B. Pro, Ser, Leu, Phe
C. Phe, Ser, Leu, Pro
D. Phe, Leu, Ser, Pro
E. Phe, Leu=Ser, Pro
E
Which of the following best describes the degeneracy of the genetic code?
A. Some codons are specified by multiple amino acids; these amino acids are usually similar in chemical properties.
B. Some amino acids are specified by multiple codons; these codons usually differ only at the first position.
C. Some amino acids are specified by multiple codons; these codons usually differ only at the second position.
D. Some amino acids are specified by multiple codons; these codons usually differ only at the third position.
E. Some amino acids are specified by multiple codons; these codons are usually unrelated in sequence.
D
Note 
Flashcard (127)