Lecture 6 Flashcards
Why is mRNA SPLICING required
It removes the introns (non coding regions)
Other words for introns
Intervening sequence
Non coding
What is exon and some other words
They the coding region of mRNA
Expressed sequence
What are the 2 methods of RNA splicing
1- Spliceosome mechanism
2- self splicing or autosplicing
What is a spliceosome?
Spliceosome is a complex protein that helps in mRNA splicing via a Spliceosome mechanism
What is a spliceosome made of
1- SnRNA( ribozymes /small nucleur RNA) + U proteins = snRNP (small nuclear ribonucleur protein / core)
2- SnRNP + other proteins + primary transcript = spliceosome
What does the ribozyme in spliceosome do
The RNA component of ribozyme (snRNA) within snRNP binds to the mRNA
The enzyme component of ribozyme within snRNP catalyses the splicing( by catalyzing the base pairing)
What do other proteins bound to snRNP do
They hold the assembly together
What does U proteins in spliceosome do ?
- U protein Recognize each other and bind to substrate ( consensus sequence of the intron) by base pairing , using their snRNA
- sequence identification
- cutting of introns
- catalyze ligation of exons together
Overview of mRNA splicing
1- SnRNPS + other proteins and primary transcript form the spliceosome
2- Splicesome cuts the intron
3- Leading to products - spliceosome components , mRNA exons bind together by ligase and cut out intron loop
What is a splice site
A splice sites are the position where spliceosome is going to cut .
These area specific sequence in pre mRNA at the beginning 5’ and end of 3’
What does snRNP in spliceosome do ?
The snRNA in snRNP binds to the splice sites
Its the snRNP molecule within spliceosome that carries out RNA SPLICING
Many snRNP ( usually labelled u1, 2, 6) carry out splicing
What is the enzyme and substrate in RNA splicing
The ribozyme (snRNA ) in the protein complex snRNP ( Us)
EXPLAIN in DETAIL the INTRON SPLICING
1- breaks ( A) are introduced at the 5’ and 3’ ends
2- sequence between exon - intron are highly conserved
3- ( exon) AG/G (intron) at 5’ and (intron) AG/G (exon)
4- these sequence are recognized by U proteins in snRNP
5- U1 snRNP Binds to the 5’ side of splice site on intron and base pairs using snRNA ( the riboenzyme catalyses the base pairing) to the pre- mRNA substrate
6- Similarly U2 snRNP binds to the branch point ( A)
7- U4/U6 and U5 bind and displace U1 - spliceosome is assemling , loop is forming
8- U4 is displaced and U6 now binds to U2
9- U5 holds the ends together while U6 carries out its job
- U6 cuts 5’ and then 3’ and joins the exon together and release the intron(lariat)
Function of U6
Cuts 5’
Cuts 4’
Joins the cut exon ends together
Release the intron
Function of U5
Holds the 2 ends together while U6 does the job
Basic properties of mature mRNA
- each code for a specific polypeptide
- found in the cytoplasm
- Attach to ribosome when translated
- only coding region translated
- most have non coding region eg 5’UTR and 3’UTR
- 5’ cap and 3’ tail in mature eukaryotes mRNA
Where is open reading frame OPR found
In the coding region
What is OPR
Its a continuous stretch of codons that code for the polypeptide primary structure
What is the function of intron
1- have specific DNA elements for bonding of TF
2- Reg sequence found in them, however not all of them
3- derive RNA splicing
4- Alternate splicing possible due to introns
How is alternate splicing helpfull
We should know that 1 gene does not equal 1 polypeptide/ protein
1 gene can actually make many polypeptide depending on the combination of exons
For example , human cells have 22,000 genes but 100,000 different polypeptides are found
Due to alternate splicing
What is Alternative intron splicing?
- processing of identical transcript in different cells can lead to mature mRNAs with different combinations of exons thus different polypeptide/proteins formed
- 95% of genes in humans undergo Alternate intron splicing
- less common in other animals and rare in plants
How do cells achieve alternative intron splicing?
- exons can be skipped or included
Skipped - some splice sites are weak and cannot attract the U protein to form spliceosome so thay exons can get omitted
Included/ retained
- some gnes express different splicing enhancer proteins ( these genes/ tissues are called specific expression)
Whichever exon it binds to will retain otherwise it would be removed
Eg it wont bind to mutated exons
Examples of Alternative Splicing
CT / CGRP
INTRON SPLICING FOR CALCITONIN. INTRON SPLICING FOR CGRP
- forms CT mature mRNA. - forms GCRP mature mRNA
- In thyroid cells. - in neural cells
- 1, 2, 3, 4 expressed. - 1, 2, 3, 5, 6 expressed
What is a genetic code?
Genetic code is a triplet codon
How do 4 bases code for 20 amino acids - how is genetic code read
- Genetic code is a triplet codom - 4to the power of 3=64 total codons
3 are stop codon( non sense) the rest are sense codon - continuous , no gaps
- no overlapping
- code is universal shared by simplest bacteria to complex humans
- code is degenerate
- redundant
What does degenrate and redundancy of geneticcode mean
Degenerate means that 1 AA has more then 1 codons
Redundancy means that 1 AA has more then 1 codons and therefore these multiple codon has same function
Use / purpose of degenerate and redundancy of genetic code
1- when a gene (base) is mutated in the third position of a codon , it results in the same amino acid being incorporated into a protein - SILENT MUTATIONS
THEREFORE , reduces risk of mutations
Two types of mutations while translating
1- synonymous (silent) mutations
2- non synonymous mutations
Explain non synonymous mutations
- mutation in the base of 1st or 2nd position of a codon , makes a different protein due to a different or no AA being incorporated
The protein may or may not be functional
All have same genetic code except
Mitochondria
Cytoplasm
Structure of tRNA
- short RNA strands of 73 to 93 nucleotides , transcribed from DNA to RNA by RNA polymerase III
- Has internal complimentarily results in 3D structure
- 4 short double helix with 3 loops form a 2D cloverleaf shape
- tertiary structure gives 3D L shape
Nucleotide
- some modified to stabilize the tertiary structure
- the 3 sequence nucleotides ( anticodon ) in the middle loops helps in the the recognition if the codon on mRNA
- some nucleotides methylated after TRANSCRIPTION
- 3’ CCA seq at the top - amino acid attachment site , helps pick the right amino acid
Function of nucleotides in tRNA
Nucleotide
- some modified to stabilize the tertiary structure
- the 3 sequence nucleotides ( anticodon ) in the middle loops helps in the the recognition if the codon on mRNA
- some nucleotides methylated after TRANSCRIPTION
- 3’ CCA seq at the top - amino acid attachment site , helps pick the right amino acid
Activation of tRNA
- tRNA is activated by the addition of the right amino acid
- this process requires energy
- this process is catalyzed by aminoacyl - tRNA synthetases (aaRS / pirate)
- every amino acod has a specofoc aaRS
- recognizes all tRNAs for the specific amino acid
- recognizes the unique part of tRNAS
- attches the right amino acid to the CCA end
- requires two high energy phosphate bonds - atp to amp
- the bond formed between aa cooh ( oh ) and CCA is a high energy bond
- the whole complex is called aminoacyl tRNA or chanrged tRNA
Function of aaRS
- recognizes all tRNAs for the specific amino acid
- recognizes the unique part of tRNAS
- attches the right amino acid to the CCA end
Which reaction consumes the most energy
Activation of tRNA
Imp info
Many of the subsequent reactions to produce a polypeptide within a ribosome are genetically favoured
How many codons for leucine
Six
How many trnas for leusine ? Why
4
Because 1 trna can bind to more then 1 codon
How can 1 tRNA bind to more then 1 codom
Due to WOBBLE HYPOTHESIS
Due to INOSINE BASE
What is wobble hypothesis
That the base pairing of the first two positions of anticodon and codon should,ld be exact however the third base isn’t supposed to match strictly
Inosine
A base on anticodon that can base pair with any base
Relationship between number of AA, codons , tRNAS
There are 20 aminoacids There are 61 codons There are less then 61 tRNA - specifically/ majorly due to wobble hypo Because 1 amino acid can have more then 1 codon And 1 tRNA can bind to-more then 1 codon
Characteristics of ribosomes
Translational machinery components Non - specific 4 rRNA and 80 small proteins Most abundant Free or attache to ER in cytoplasm
Whee does rRNA transcription occurs?
In the nucleolus
rRNA is transcribes in the nucleolus
Ribosomes are produced in the nucleolus
How many copies of rRNA are there
200 in the genome
These copies are in 5 clusters in humans called
Nucleolar organizer regions
What happens in nuclelar organizer gene
rRNA is transcribed
Ribosomal subunit made using ribosomal proteins
Meaning ribosomes made here
Ribosomal subunits assembled here
Then is transported to cytoplasm via nuclear pore
rRNA transcription
The sequence and structure of all rRNA gene is identical
Many RNA POL I transcribe the gene forming a Christmas tree
The initial transcript is called the 45S transcript that contains 18S 5.8S and 28S rRNAs
Some are catalytic and some are structural.
