Study BMB 400 Chapter 13 Flash Cards

 
Pile Management Card
BMB 400 Chapter 13

loading
tRNA nucleotidyl transferase
-in euks, CCA is added after transcription
how to add CCA to tRNA?
euks: tRNA nucleotidyl transferase adds CCA AFTER transcription

pros: CCA is encoded in the DNA
pre-tRNA introns
-very short (about 15 nts)
-no consensus sequences
-removed:

-Cleavage by an endonuclease
-Phosphodiesterase to open a cyclic intermediate and provide a 3’OH
-Activation of one end by a kinase (with ATP hydrolysis)
-Ligation of the ends (with ATP hydrolysis)
-Phosphatase to remove the extra phosphate on the 2’OH (remaining after phosphodiesterase )
RNase D
Exonuclease trims 3’ to 5’, leaving the mature 3’ end
RNase F
Endonuclease cleaves 3 nucleotides past the mature 3’ end
RNase P
Endonuclease cleaves to generate the 5’ end
RNase III
-no apparent primary sequence specificity
-stems of stem-loops
Cutting and Trimming RNA
-endonucleases to cut at specific sites within a longer precursor RNA
-exonucleases cut at ends to make mature products
-in pro and euks for ALL RNA
types of RNA processing
A) Cutting and trimming
B) Covalent modification
C) Splicing
nuclear pore complex
-how mRNA gets out of nucleus
-proteins associated with the RNA carry nuclear export signals
-GTPase Ran hydrolizes GTP to get energy
Guide RNA-directed uridine insertion or deletion
-Part of the guide RNA is complementary to the mRNA in vicinity of editing (Fig. 13-26).
-Uses a guide RNA (in 20S RNP = editosome) that is encoded elsewhere in the genome
Site-specific deamination
-only in certain tissues
-regulated manner
-Cytidine deaminase converts the C to U
-ADAR (adenosine deaminase acting on RNA) converts Adenosine to Inosine. Inosine can base-pair with cytosine.
Methods of RNA Editing
1) Site-specific deamination
2) Guide RNA-directed uridine insertion or deletion
RNA editing
-changes RNA sequence after transcription
-as much as 55% of the nucleotide sequence added after transcription
-can add, delete or change nucleotides
Exon shuffling advantage
- exons usually encodes an independently folding unit of protein
-different proteins via exon duplication and divergence
-extra mileage: related exons are sometimes in unrelated genes
-Exons (~150 nt) while introns up to several hundred kb); rcombination is more likely to occur within the introns that within the exons
The intron advantage
- regulate gene expression via alternative splicing
-produce multiple products from a single gene.
-Create new genes by reshuffling exons
introns late model
-introns were inserted into genes by a transposon-like mechanism.
Intron early model
-introns were removed from pros because of a selective pressure to increase the rate of chromosome replication and cell division.
Regulated alt splicing
-repressor protein can overcome repressor site
-activator protein can splicing enhancer to not translate that region
if intron is not removed from mRNA
-no transport
-no protein synthesis
if an exon contains a stop codon
-truncated protein
SR proteins
-bind RNA using RNA-recognition motif
-recruits splicing machinery using RS domain
Repressor
exonic splicing silencers (ESS)
-mostly heterogeneous nuclear ribonucleoprotein (hnRNP)
-bind RNA but lack the RS domains
-can't recruit the splicing machinery
Enhancer
exonic splicing enhancers (ESE)
Alternative splicing
-switches gene expression
SV40 T antigen
-ratio of 2 proteins produced (T-ag vs. t-ag) depends on level of SF2/ASF (SR protein)
-if a lot of SF2/ASF, produces t-ag
alt splicing occurs in...
...all metazoa
...a lot in vertebrates
...60% of human structural genes are subject in alternative splicing
products of alternative splicing
-constitutive or regulated
-what spliced depends on cell type, conditions, tissue, etc.
minor spliceosomes
-in higher euks (mammals, plants)
-alternate spliceosome
-aka AT-AC
-recognizes rare introns that contain consensus sequence distinct of the most pre-mRNA introns.
-some components same, some different to recognize different introns
-same chemical pathway, though
SR proteins recruit
U2AF to 3' splice site
U1 to 5' splice site
exon splicing enhancers
-SR (Serine Argenine rich) proteins bind to exonic splicing enhancers (ESEs) with the exon
-SR proteins recruit spliceosomes to nearby splice site
-specially U2AF to the 3’ splice site and U1 snRNP to the 5’ site
-SR proteins essential for splicing
Two ways to enhance splicing accuracy
1. Coupling between RNA transcription and splicing
2. Use exonic splicing enhancers
What genes lack introns in higher euks?
histones and interferons
Gene splicing
-introns rare in pros, yeast
-introns more in higher euks
-introns constitute ~80% of typical vertebrate structural genes
Why aren't Group II and I RNAs not enzymes?
-b/c turnover= 1!
-to turn into enzyme, need free G and complementary IGS sequence
-can change IGS to cleave whatever RNA we want
RNA evolution
-Before, many catalytic functions in the pre-mRNA splicing may carried out by RNAs
-these functions now performed by proteins
-now, catalytic center is still formed solely by RNA
-but proteins still do their job
Noble prize
-Additional proteins are NOT needed for splicing of pre-rRNA!

-just need GMP, GDP, GTP or Guanosine
enzyme RNAs
Ribonuclease P (RNase P): Cutting and trimming to generate ends of rRNA, tRNA and mRNA
Group I introns
Group II introns
snRNAs in splisosome involved in splicing
Hammerhead ribozymes: cleavage
rRNA in ribosome: peptide bond formation
internal guide sequence
IGS determines 5' splice site
Group I intron shape is...
...linear
-ribose G nucleotide for RNA cleavage
-binds to G pocket
-Internal guide sequence (IGS)
-b/w 400-1,000 nts long and much of sequence important for folding/catalysis
-High [G] in vivo prevents reverse reaction
self-splicing introns
-intron folds upon itself into a specific conformation to catalyze own release
-Group II= lariat
-Group I= linear intron
Group I introns
-rare
-some organelles in euks, some pros
-branch site G
-RNA enzyme encoded by intron (self-splicing)
Group II introns
-rare
-mito, chloro (euk organelle introns)
-branch site A
-RNA enzyme encoded by intron (self-splicing)
nuclear pre-mRNA introns
-euks
-very common
-branch site A
-major/minor spliceosomes
3 types of introns
nuclear pre-mRNA
Group II
Group I
C complex
U1, U4 leave
-triggers catalysis
-lariat half-formed
B complex
-all 5 U's bind
A complex
U1, U2 bind
different spliceosome proteins can bind to same sequence
ex: Same sequence is recognized by a protein (BBP) at one stage and displaced by snRNP U2 at another
spliceosome assembly
1. U1 snRNP binds (and base pairs) to the 5’ splice site.
2. BBP (branch-point binding protein) binds to the branch site
3. U2 snRNP binds (and base pairs) to the branch point, BBP dissociates
4. U4U5U6 snRNP binds
5. U1 snRNP dissociates
6. U4 snRNP dissociates

-all requires ATP hydrolysis
snRNPs
-U1, U2, U4, U5 and U6
-of spliceosome
-recognize the 5’ splice site and the branch site
-bring those sites together
snRNAs
-U1, U2, U4, U5 and U6
-in spliceosome
-100-300 nts long
-assembled w/ proteins (snRNPs)
Spliceosome machinery
-to splice exons!
-150 proteins and 5 RNAs, size is similar to a ribosome
-many functions expressed by RNA components
trans-splicing
-splice 2 different RNAs together
-Y shaped lariats
-in C. elegance and higher euks
-rare, though
-chemistry of trans and cis the same
why does splicing go forward?
1. increase in entropy
2. intron rapidly degraded (no 5' cap or polyA tail)
spliceosome energy
-needs ATP for assembly/use
-none for breaking/making bonds
RNA splicing
-no extra energy required
-2 phosphodiester bonds formed and broken
-only ATP needed to operate spliceosome
to remove intron....
2 transesterification rxns:
1. 2’ OH of the conserved A at the branch site attacks phosphoryl group of the conserved G in the 5’ splice site. It forms three-way junction.
2. Newly formed 3’OH of 5’ exon attacks the phosphoryl group at the 3’ splice site
-joins the 5’ and 3’ exons
-release “lariat”
AG
-3' splice site
GU
-5' splice site
Branch point site
found entirely within the intron, usually close to its 3’ end, A followed by polypyrimidine track
important splice sites
-5’ splice site: 5’ end of the intron: GU
3’ splice site: 3’ end of the intron: AG
Branch point site
# introns per gene
-goes up higher on the animal chain
Place this card into pile: