Final Exam Flashcards
RNA processing
Governs how, when, and whether RNA will be used
in a cell
Primary Transcripts
- All eukaryotic RNAs and some Bacterial RNAs are synthesized as biologically inactive precursors that must be chemically modified during or immediately following transcription
- Cleavage and/or joining of RNA strands and sometimes modification
- Errors in splicing can lead to problems like cystic fibrosis and spinal muscular atrophy
*Once an mRNA transcript has reached the end of its utility in the cell, it is subject to a final type of RNA processing—degradation. Specific enzymes remove the 5′ cap and the 3′ poly(A) tail, and the rest of the RNA is broken down by cleavage of the phosphodiester backbone.
RNA processing is catalyzed by
RNA (splicesome) and Proteins
-different locations in bacteria vs eukaryotes
mRNA RNA processing occurs in?
- In the nucleus
- Post-processing, mRNA is exported to the
cytoplasm
what are the 3 major modifications that occur during mRNA processing?
- 5’ end gets a 7-Methylguanylate cap (protective)
- 3’ end gets a poly A tail 100-250 nt long
- Introns are spliced out
- Regular splicing
- Alternative splicing
What is responsible for the 3 major modifications in mRNA processing
- Enzymes responsible for these three events do not operate independently, they work together
- Processing is also coupled to transport from the nucleus to the cytoplasm
Describe the steps to get from DNA to mRNA
1.Transcription, 5’ capping
2. Endonuclease causes cleavage at the poly-A site
3.Poly A polymerase(PAP) + ATP do polyadenylation
4.RNA splicing occurs
RNA capping has what type of bond
- 5’,5’ triphosphate linkage
What is the name of the capping enzyme?
guanylyltransferase
Describe how capping occurs and why it’s important.
Only RNAs made by RNA pol II are capped because the mechanism is specific to the C terminus of the RNA polymerase
* As the nascent RNA emerges, at about 20-30 nt into the process, capping occurs
—This is one mark of RNA as an mRNA
* crucial for binding of the mRNA to the ribosome during translation
In experiments what happens when the 5’ cap isn’t present?
The 5′ cap requirement for ribosome binding and translation., if the 5’ is missing it is seen that all mRNA is unbound
How long is the Poly A tail and why is it significant to mRNA
- PolyA tail is about 80-250 nt long
- 3’polyadenylated mRNAs survive much longer because they are protected from exonuclease activity through binding of protective enzymes
How is the poly-A tail added?
- PolyA tail is added in multiple steps involving polyadenylation factors, polyadenylate polymerase (PAP), and poly(A) binding protein PABP
- The first thing that happens is that Pol II extends the transcript beyond the site where the polyA tail is to be added
- Cleavage happens at the polyA addition site by an endonuclease enzyme associated with the C terminus of the Pol II enzyme
Polyadenyoaton factors are associated with?
Pol 2 during transcription
mRNA capping polyadenylation and splicing are coordinated by
- All about the association with Pol II
- The C terminus of the Pol II proteins associates
with factors involved in these processes - RNA processing can therefore basically occur simultaneously with transcription
- This leads to rapid processing and production of mature mRNAs
What does the Splicing and editing do for the proteome
-both splicing and editing expand the coding capacity of the genome by creating mRNA that is directly encoded by the DNA
-Proteome shows that in any case the number of different proteins greatly exceeds the number of identified genes.
What is splicing and what are some of the aspect due to splicing?
- Splicing (does not occur in bacteria)
-Ordered breaking and joining of specific phosphodiester bonds to - Splicing also prepares mRNAs to be recognized by proteins that export them from the nucleus and promote their translation by the ribosome.
achieve the precise excision of introns - Requires accurate base pairing between pre-mRNA and splicing machinery
- Spliceosome is responsible for most splicing
- Small number of introns found in mitochondria, chloroplast, and bacteriophage are self-splicing
Describe introns
- In vertebrates, the vast majority of genes contain introns
- Histone genes are an absolute exception
- Most genes in simpler eukaryotes like yeast, lack
introns but some have them - Very rare but do occur in bacteria and archaea
- Very rare but do occur in some bacteriophage
- Introns seem to be important for the amount of mature mRNA that is produced
- Regulatory small RNAs for example come from introns often
- Are transcribed as part of the transcription process
- Size 50-20000 nucleotides while exons are 100- 1000 nt
what did the DNA-RNA hybrid reveal
the presence of introns
what is the typical intron length in mammals
pre-mRNA includes eight introns with an aggregate length 5 to 10 times that of the flanking exons.
What is Alternative splicing?
Process in which exons in the primary transcript from a single gene are spliced together in various combinations to produce different mRNAs and thus different polypeptides
Describe some of the aspects seen due to alternative splicing
When we compare an organism’s genome to its proteome, in many cases, the number of different proteins greatly exceeds the number of different genes
* More than 90% of human genes undergo alternative splicing
* Certain exons are selected for inclusions and others are not, but the order of the exons does not change relative to the primary transcript
* Many mammalian genes have 2 or more alternatively spliced mRNAs (splice variants)
How does alternative splicing work?
- We don’t fully understand it
- Splice sites—nucleotide sequences within the intron and at the borders between introns and exons seem to be important
-Sometimes these sequences can be masked on purpose
Why is alternative splicing important?
- Alternative splicing can be important for human disease
-CD44 which is a cell surface receptor
— Inclusion of a specific exon in the spliced mRNAs is associated with the progression of certain tumors from localized to invasive growth
— Thus, understanding what determines exon choice during alternative splicing might lead to new therapeutic strategies to treat or prevent some cancers.
Splicesome and splicing have key parts that are conserved which are?
5’ splice site contains the GU
Branch point which is an A
3’ splice site contains AG
Describe the steps that occur during splicing without the proteins involved
1.The brain-points 2’OH attacks the 5’splice site
2. The 5’ splice site is now activated to attack the 3’ splice site
3. The intro is released from the spliced mRNA as a lariat
When splicing occurs what are the reactions occurring and what are the by-products from the reaction?
First transesterification and second transesterification
with spliced exons and excise lariat intron
snRNP’s
- Splicesome
- 5 small nuclear ribonucleoproteins + hundreds of additional
proteins - Each snRNP has a single small nuclear RNAs (snRNA)
-These snRNAs are very conserved - U1, U2, U4, U5, U6 snRNAs range in length 107-210 nucleotides
- Associated with 6-10 proteins
- There is base pairing between pre-mRNAs and snRNAs
Why is base pairing more important than exact nucleotide in of snRNPs with pre-mRNA?
improper base paring leads to blocking of splicing
Describe splicing with the presence of proteins
- U1 binds to the 5’ splice site and U2 binds to the branch point
- The U4-U6-U5 trimeric snRNP displace U1 at the 5’splice site, then U4 dissociates
- U6 and U2 catalyze attack of the branch point on the 5’ splice site
- the 5’ splice site attacks the 3 splice site completing the reaction
Why are U1 and U2 keys in splicing
U1 and U2 allow for the first transesterification to occur allowing splicing to commence
self-splicing was discovered by who and when?
Discovered by Tom Cech in the 1980s in Tetrahymena
Describe aspects of self-splicing
-400-1000nt
-group 1 and 2 introns
- found mostly in bacteria organelles and fungi
-do not need proteins to self-splice in vitro
Is self-splicing really able to do the work on its own?
It is important to note that group I and group II introns do require proteins for splicing in vivo—not for catalysis, but for forcing the pre-RNA into the correct conformation for splicing to occur.
RNA editing
- Change the sequence of an RNA after it has been transcribed
- Compare RNA sequence with genomic sequence because the edit is not happening at the genomic DNA level
- Substitution of bases
- Insertion or Deletion of bases (using guide RNAs)
- Editosome (16 proteins)
Editosome
- Complex of 16 proteins
- Guide RNA that are partially complementary to the pre-mRNA regions that will be changed (35-75 nt)
- U nucleotides are removed and added
- Substitutions involves deamination of A or C by enzyme called ADAR
cytidine deaminase
converts C to U
Describe one example of RNA editing causing changes of the same mRNA.
ApoB carries bad cholesterol “LDL”
Different versions of ApoB—affect the way cholesterol is metabolized
changing one nucleotide can encode C into a U and lead to a translated protein that is much shorter than the full-length protein
What organisms share the same genetic code with a few exceptions?
bacteria, yeast, amphibians, plants archaea, and humans
why is it significant that all organisms share a genetic code
the universality of the genetic code provides amazing strong, molecular evidence for evolution , much more compelling than argument based on body shapes and the fossil record
How many different nucleotides
4 different nucleoties
how many nucleotides combine to create the genetic code of proteins
combination of 3 nucleotides ( this produces possible 64 code words)
a combination of two wouldn’t be enough only making 16 code words
Genetic code aspects
DNA triplets to amino acids
-20 amino acids
-64 possible codons ( some specifically the same amino acid)
-61 specify amino acids
-3 specify stop
-methionine ( AUG, start) and tryptophan only have one codon
-Leucine, serine , arginine specified by six different codons
Explain how codon families are structured
the first two nucleotides are the same, the nucleotide at the third position does not matter and the base paring of the first 2 nucleotides is key
Name some of the exceptions to the universal code
UGA - Normally stop but can code for TRP
CUG - Normally Leu but can code for Thr
UAA, UAG- Normally stop but can code for Gln
UGA- Normally stop but can code for cys
tRNA hypothesized by who
- In 1955 Crick hypothesized the existence of an adaptor molecule that can recognize codons on the mRNA and couple that with amino acids
Describe the componets that make up the structure of tRNA
- Cloverleaf
- 70-90 nucleotides long (smaller in mitochondria and chloroplasts)
- Free 3’ and 5’ ends
- @3’ end ACC (added during processing)
- Anticodon is at the center of the middle loop: codon-anticodon base pairing
- Post-transcriptional: modification of nucleotides
- When tRNA has an amino acid, it is called aminoacylated (at the 3’end)
ACC exists on which end of the tRNA
3’ end which is the acceptor stem
what are the unusual nucleotides that exist in tRNA
dihydrouridine, inosine, ribothymidine, pseudouridine
what are the components of the tRNA
acceptor stem, D loop. TYCG loop and the anticodon loop
what major component does the anticodon loop contain?
the anticodons which antiparallel bind to the mRNA codons
Inosine can form bonds with
C,U,A
Amino acids bond to which end of the tRNA
amino acid arm which is in the acceptor stem with the ACC
True or False all tRNA fold up into a precise 3D structure base paring is antiparallel.
true
Describe some of the codon-anticodon interactions
- One tRNA can recognize more than one codon corresponding to a given amino acid
- The5’firstbaseoftRNAbase pairing with the 3’ third position is called WOBBLE
—- Can form non-standard base pairing at this particular position (non-WC pairing)
——GU
——-Inosine (deaminated adenosine) can bind to A, C and U
who proposed the wobble hypothesis
The process by which some tRNAs can recognize more than one codon was formalized by Crick, who proposed the wobble hypothesis
Which bases pairs always watson-crick base pair
The first two bases of an mRNA codon always form Watson-Crick base pairs with the corresponding bases of the tRNA anticodon, and they confer most of the coding specificity.
what is the minimum amount of tRNAs for coding all 61 codons?
A minimum of 32 tRNAs are required to translate all 61 codons (31 tRNAs for the amino acids and 1 for initiation).
position 1 is C in tRNA
pairs with G
position 1 is A in tRNA
pairs with U
Position 1 is G in tRNA
pairs with C and U
Position 1 is U in tRNA
pairs with A and G
Position 1 is I in tRNA
pairs with C , A , and U
ribosomes read mRNA in which direction
5’-3’ without gaps
Reading frame plays what role in translation
- Depending on where you start translation, you can have different reading frames
- There are initiation codons and termination codons
- Open reading frame or CDS encodes a protein
name for amino acid attached to tRNA
aminoacyl-tRNA
name of the enzyme that binds amino acid to tRNA
aminoacyl-tRNA synthetase
Attaching amino acid to transfer RNA requires
- Reaction occurs in the cytosol and not on
the ribosome - ATP hydrolysis is used
- There is a specific synthetase for each amino acid
- All synthases need magnesium
- Most organisms have 20 different
synthetases - It has built-in proofreading ability
Mistakes in translation are 1 in 50,000 codons - Ribosomes cannot discriminate between correctly or incorrectly charged tRNAs
Huge responsibility for this enzyme
What end do amino acids get added to?
Amino acid is added to the 3’ end of the tRNA onto the 3’ adenosine reacting with the carboxyl-terminal of the amino acid
-When attached to an amino acid, the tRNA is charged
the attachment of amino acids occurs in how many steps
The reaction occurs in two steps
* Step 1: Adenylylation
* Step 2: tRNA charging
How many classes of synthetase exist?
There are two classes of Synthetases, Class I and Class II
Class I, add aa onto the 2’ OH
Class II, add aa onto the 3’ OH of tRNA
Adenylyation
the process which an Amino acid reacts with ATP which the releases PPi and forms Aminoacly-AMP
tRNA charging
a process that sees the 3’ end of tRNA to attack the C terminus of the amino acid either by the 2’OH or the 3’OH which is called synthetases.
Trasnesterficaiton
when the amioacyl-tRNA goes from 2’OH attachemnt to a 3’OH attachment
which class of synthetase is larger?
Class 2
Is each synthase specific to one amino acid?
yes but can recognize more than one tRNA most aa are specified by more than one codon
what is the second genetic code?
interaction between the aminoacyl-tRNA synthase and tRNA (critical role in maintaining the accuracy of protein synthesis)
Who discovered the ribosome?
1974 George Palade ( received Nobel prize for it)
What was the ribosome originally thought to be?
Organelle but was not encapsulated by lipids
Is the ribosome evolutionarily conserved?
true
how many ribosomes are in bacterial cells
15,000 ribosomes ( 25% of dry weight)
The function of ribosomes?
-bring together mRNA codon with tRNA adaptor
-catalyze peptide bond formation
Describe some of the components of ribosomes
-complex of rRNA and protein
-highly conserved between species
-S complex refer to sedimentation rates
-3 or 4 rRNA subunits
-dissimilar sequences but structure is conserved
-up to 83 proteins
Ribosomes in Prok
50s + 30s =
70s
Ribosomes in Euk
60s + 40s =
80s
What makes up 50s in E coli?
5s rRNA and 23S rRNA
What makes up 30s in E coli?
16sRNA
Name the 2 big components that make up prokaryotic 70s ribosomes
23s(2900rnt) + 16s(1500rnt)
Name the 2 big components that make up eukaryotic 80s ribosomes
28S:5.8S(4800rnt ,160rnt) +18S(1900rnt)
Which components of the ribosomal subunit are involved in catalyzing peptide bond formation in all organisms?
rRNA
Describe the process of translation
- Activation of amino acids: the tRNA is aminoacylated
- Initiation: the mRNA and the aminoacylated tRNA bind to the small ribosomal subunit the large subunit then binds as well
- Elongation: successive cycles of aminoacyl-tRNA bind and peptide bond formation occur until the ribosome reaches a stop codon
- Termination: translation stops when a stop codon is encountered. the mRNA and protein dissociate and the ribosomal subunit is recycled
- Protein Folding
The protein exit tunnel is adjacent to which site?
the P site and only wide enough to allow the alpha helix or unfolded polypeptide to pass through
What are the 3 sites of biding in ribosomes
E, P, A
Explain what occurs in initiation for translation
-Small subunit of the ribosome must be recruited to the mRNA
-identification of the initiation codon
-Association of charged initiator tRNA with the mRNA
-Recruitment of the large ribosomal subunit to form the active ribosome
What are the names of the factors needed for initiation
-IF in bacteria
-eIF in eukayotes
Where does aminoacyl-tRNA position itself during initiation?
The P site
How does the recruitment of ribosomes occur in prokaryotes?
-ORF has ribosome binding sites(RBS) (shine-Delgarno sequences) 3-9 bases on the 5’ side of the start codon complementary to 16s rRNA 3’ end; this helps align the ribosome
-GGAGG
How does the recruitment of ribosomes occur in Eukaryotes?
-5’cap:binds elF4e(cap-binding protein)
-Kozak sequences
interact with initiator tRNA and rRNA
-poly A tail at 3’end
Efficient recycling of the ribosomes(Foster reinitiation)
Protein synthesis begins at which terminal?
N terminal
the 5’ AUG specifies an N terminal methionine
All organisms have two different tRNA for methionine what are their functions?
-one exclusively for intiation
-one for general use (internal methionine
-initiation factor binds to the initiation tRNA
What are the differences of E coli and Eukaryotes in the activation of amino acids?
None
they share all these
-20 amino acids
-20amicoacyl-tRNA synthetases
-32 or more tRNAs
-ATP
-Mg2+
Name the similarities and differences of E coli and Eukaryotes in Initiation.
Shared
-mRNA
-Start codon
-GTP
-Mg+2
Differences
-E.coli: N-Formlumethionly-tRNA fmet
EUk has methionyl-tRNA met
-Ecoli has 30s and 50 s
Euk has 40s and 60s
-E coli has 4 IF
Euk has many eIF
Name the similarities and differences of E coli and Eukaryotes in Elongation
Shared
-GTP
-Mg+2
Differences
-E coli 70s ribosome (initiation complex
EUk 80s (initiation complex)
-E coli has EF
Euk has eEF
Name the similarities and differences of E coli and Eukaryotes in Termination and release
Shared
-Stop codon in mRNA
diffrences
-E.coli has RF
EUk has eRF
Tranlation initiation occurs usually at which point?
the first AUG from the 5’ end of the mRNA
What determines the start vs. normal
-two different methionine tRNAs
-same synthase charges both
-only methionyl-tRNAiMET binds to the P site (met is also used as a regular aa tRNA is different during initiation)
Specific steps for Initiation
1.The 30s subunit bind IF-1 in the E site and the IF-3 to the A site . The mRNA is then bound
2. the fMet-tRNA accompanied by the IF-2(bound with GTP) then base pairs with the start codon which is in the P site.
3. The 50s subunit associated with the 30s unit. which leads to the release of GDP, Pi, IF-1,2,3 this now has formed the 70s initiation complex
Describe the characteristics of elongation in bacteria
● Entry of succeeding (next) aminoacyl-tRNA (in the A site)
● Formation of a peptide bond (between A and P sites)
● Movement of the ribosome one codon at a time along the mRNA ● Uncharged tRNA moved to the E site for exit
● Involves EF (elongation factors): EF-Tu, EF-Ts, EF-G
● GTP hydrolysis facilitates binding of incoming aminoacyl-tRNA and the movement of the ribosome along the mRNA
Steps for elongation
- Binding of second aminoacyl-tRNA at the A site
- Peptide bond formation between the amino acid in the P site and amino acid in the A site, transferring the growing polypeptide to the tRNA in the A site
- Translocation; resolution of the hybrid binding state by shifting the tRNAs and mRNA by one codon
Describe STEP 1 of elongation in more detail
- EF-Tu-GTP helps bind appropriate incoming aminoacyl-tRNA
- GTP converted to GDP which releases EF-Tu-GDP (this is regenerated in a process that involves EF-Ts)
- This step allows some time for proofreading of the codon- anticodon interaction
Describe STEP 2 of elongation in more detail
-Peptide bond formation between the tRNAs in A and P sites on the ribosome: Peptidyl transferase reaction
-Amino acid chain is formed on the tRNA located in the A site
-Peptide bond formation is catalyzed by 23S rRNA of the large ribosomal subunit
-At least 4 GTPs must be broken to generate each peptide bond
What is the name of the reaction to attach the amino acids in elongation?
peptidyl transferase reaction
Do peptide bonds happen between? (elongation)
Two charged species
-peptidyl-tRNA + aminoacyl-tRNA
-peptidyl transferase reaction onto the aminoacyl tRNA
( reaction between tRNA in the P site and A site)
Describe STEP 3 of elongation in more detail
Peptidyl-tRNA must move from the A to the P site
* Translocation involving a change in ribosomal conformation resulting in movement along the ribosome
* EF-G-GTP binds to the A site because of GTP hydrolysis and displacement of the A-site peptidyl-tRNA
* Eventually EF-G-GDP is released making the A site available again
Termination is signaled by
stop codon
What must occur for termination to occur in bacteria
● Hydrolysis of the terminal peptidyl- tRNA bound
● Release of the free polypeptide and the last tRNA now uncharged from the P site
● Dissociation of the 70S ribosome into 30S and 50S subunits
What is the role of the release factors in termination?
- RF-1 (recognizes UAG and UAA), RF-2 (recognizes UGA and UAA), RF-3-GTP when bound directly to ribosome and RF-3-GDP when bound to RF-1 or RF-2
- Recycling of Ribosome RRF and EF-G separate the ribosome from the mRNA and tRNA
Describe the process of termination with the RF role.
RF-RF3-GDP (RRF) complex binds to the stop codon in the A site of the ribosome
-this binding causes the release of the polypeptide
-EF-G-GTP complex shifts the RF factor complex and binds to the A site while also releasing Pi
-GTP hydrolysis occurs leading to the dissociating of the EFG-GTP, RRF , and ribosomal subunits
-In the Final step IF-3 then binds to the 30s subunit of the ribosome ready to intimate the cycle again.
Describe the role of antibiotics with translation.
- 40% of antibiotics inhibit translation
- Bind translation apparatus at different steps
●Tetracycline= binds 30s subunit; inhibits aminoacyl-tRNA to the A site
●Erythromycin=binds the peptide exit tunnel on the large ribosomal subunit; blocks exit of the growing polypeptide chain
