Molecular Biology Exam 2

what enzyme is found all over the nucleus?

exonuclease

what are the two major signals for RNA degradation?

5’ NTP phosphate and 3’ ribose hydroxyl

before the promoter is cleared, what is attached to the promoter region of DNA?

pre-initiation complex

after promoter clearance, what binds to DNA to begin RNA transcription?

RNA Polymerase 2

after the promoter escapes (the promoter region), what is now committed to occur?

transcription

how many nucleotides can RNA polymerase synthesize in one second?

20-30 nt

how do we make sure that RNA coming from the polymerase isn’t immediately degraded?

Caps

how do Gcaps help increase the efficiency of splicing mRNAs?

increase recognition of GCaps

what is the max number of methyl groups likely to be added to RNA?

3

what is step 1 of GCap formation?

RNA triphosphatase cuts off one phosphate group

what is the second step of GCap formation?

guanylytransferase adds guanosine to ppXpY (diphosphate)

what is the third step of GCap formation?

methyltransferase adds methyl group to 7’Gp (end of formation)

how many polypeptides is RNA polymerase?

12 polypeptides

what is the nuclear cap-binding complex?

an 80kD and 20kD protein complex that is highly conserved throughout eukaryotes that communicates with nuclear events such as splicing

what does elF4E do?

directly binds to m7G cap

what is elF4G?

a scaffolding protein that connects to the elF4E complex

what does elF4F do?

focus on translation

what does Dbp5 do?

knock off cap-binding complex which dissociates RNA helicase

what RNA mods exist in metazoa?

deamination (a → i; c → u)

what RNA mods exist in fungi?

Chytridiomycota tRNA substitution editing by insertion

what RNA mods exist in excavata?

deamination (C → u)

what RNA mods exist in archaeplastida?

deamination (c → u; u → c)

what is the purpose of ADARs (adenosine deaminases acting on RNA)

to modify certain adenosines to inosines which stops incorrect base pairing (amine → carboxyl)

what are there 4 different ADARs in humans?

different specialties for different functions

how many editing sites are there across the human transciptome?

»23,000

what can inosine change in coding?

amino acids

what does the GRIA2 gene change in the brain?

Ca2+ permeability (glutamine - G → arginine - R) and receptor desensitization (R → glycine - G)

what does the GLIA3 gene change in the brain?

receptor desensitization (R → G)

what does the APOBEC enzyme complex do to edit C to U in RNA?

cuts glycosidic bond and replaces C with U

what is the importance of changing the C to U in RNA?

can initiate new ORF which can affect encoded amino acids (stops by same mechanism)

how does editing create basepair stems in tRNA?

nucleotide insertion

how does editing create substrates for base mods in tRNA?

base conversion

how does editing create substrates for 5’ and 3’ processing?

base converstion

how can editing change tRNA identity?

base conversion in the anticodon loop

how does editing add conserved sequence elements in tRNA?

nucleotide insertion

how does editing correct stem mismatches in tRNA?

base conversion

how does editing complete the overlap of tRNA?

nucleotide insertion

how does potential modulation of translation efficiency occur in RNA?

base conversion

how does editing create conserved structural elements in RNA?

nucleotide insertion

how does editing create conserved structural elements in RNA?

base conversion and nucleotide insertion

what occurs in the body when there are improperly modified RNAs?

accumulation/destruction of RNA in an inappropriate manner

what does a Self tRNA + TLR7 create?

immunological suppresion

what does a bacterial tRNA + TLR 7 create?

immunological activation

what happens to translation when exposed to a stressor?

RNA mediates the stoppage of protein translation

how does the presence of an ADAR in RNA suppress miRNA production?

inosine cause less cleavage to occur thus decreasing the generation of miRNAs

how do gRNAs help RNA in mitochondria of trypanosomes?

direct the insertion and deletion of UMPs

what occurs when gRNA is missing an A or G to pair with U in mRNA?

U is removed

how does the U get removed from mRNA?

cutting beyond U or exonucleases or ligating the two pieces of pre-mRNA together

what happens when the mRNA is missing a U to pair with A or G?

U is added back in

where does the U come from?

UTP

what is step 1 of pre-mRNA splicing?

branch point nucleotide will break the phosphodiester bond from the 5’ splice site

what intermediate is made after the 1st step of slicing?

a lariat intermedite (loop of introns connecting back to the bp nt)

what is the 2nd step of pre-mRNA splicing?

free 5’ ss connects back to exons, releasing the lariat intron

what is the average number of introns per gene in humans?

8

what allows for humans to be able to splice so many introns?

production of ATP at faster rates

what type of cell typically has fewer introns per gene?

lower eukaryotes

how many bp make up internal exons?

145bp

what makes up the bulk of RNA polymerase 2 transcripts?

introns

how long are many primary RNA polymerase 2 transcripts?

100,000 nt

what is the longest primary transcript at 2.4 million nt?

dystrophin gene

where is the most complex gene structure present?

titin gene

after splicing the titin gene, how many nt?

80,760 nt (177 introns)

what is the longest known single exon?

17,106bp in titin gene

what two nt always start off the 5’ ss in introns?

GU

what is located in the branchpoint region?

UACUAAC box

what is located at the 3’ss in introns?

AG

what nt is known as the branch point specifically?

A

what can occur if even one nucleotide in a ss signal is mutated?

site can be skipped

in the 1st step of splicing, what gets deprotonated?

2’ hydroxyl (allows for oxygen to bond to phosphate group of exon 1 - lariat intermediate)

in the second step of splicing, what happens to the newly made 3’ hydroxyl of exon 1?

deprotonation (allows for O to bond to phosphate group of intron)

what binds to the 5’-A-3’ of the bp nt in a lariat intermediate?

2’ GU of the 5’ss

what is a snRNP?

complex of snRNA which identify base pairing of 5’ss and bp

what snRNP binds to the 5’ ss 1st?

U1

what snRNP binds to the bp sequence?

U2

what is the commitment complex?

GUAAGU - does not leave until other snRNPs arrive

what does the tri-snRNP do to complex?

bind to exon 1 (consists of U4, U5, and U6)

what snRNP is unwound from U6 by Brr2p RNA helicase?

U4

what replaces U1 after it is unwound from 5’ ssby RNA helicase Prp28p?

U6

what does helicase Prp2p (ATP independent) do?

structural rearrangement of intron followed by protein removal (ATP dependent)

what occurs after helicase Prp2p removes a protein?

exposure of 2’ OH in proximity to 5’' ss (initiation of lariat intermediate)

what does helicase Prp22p (ATP independent) do?

structural rearrangement (forms lariat intermediate)

what does helicase Prp16p (ATP dependent) do?

structural rearrangement to expose 3’OH in proximity to 3’ ss

what displaces mRNA?

RNA helicase Prp22p

why do some organisms seem to be gaining introns and some losing them?

gaining to test out evolutionarily, losing to conserve energy

why do we splice in a test tube?

easily modify pre-mRNA, selectively remove proteins, change reaction condition

what makes up 99.5% of pre-mRNA splicing events?

canonical spliceosome

what nt are located at the 5’ ss in minor introns?

AT/AU

what nt are located at the 3’ ss in minor introns?

AC

what snRNP replaces U1 in minor class?

U11

what snRNP replaces U6 and U4 in the tri-snRNP?

U6atac and U4atac

in the minor class pathway, what do U11 and U12 do in the 1st step?

they connect together

in the minor class pathway, what does the U11/12 bind to?

U11 binds to 5’ ss of exon 1 while U12 binds to bp nt A and exon 2

in the minor class pathway, what happens in the 3rd step?

U11 leaves and is replaces by tri-snRNP

in the minor class pathway, where is U5 in relation to the exons and lariat?

U5 is between exons and across from lariat which is still not cleaved

what increases as the number of genes increase?

organismal complexity

what allows for “cassette” splicing?

increased specificity choosing one exon over another

in alternative 3’ splice site usage, what happens to the exon when distalled spliced?

some of the exon can be spliced as well

which type of genes are more likely to be alternatively spliced?

developmental body pattern genes

how does a negative protein precent binding of a protein or snRNP?

prevents binding of U1 and U2, skipping full exons

how does a positive protein assist in recruiting a protein of snRNP ro the correct site?

serves as an attractant for snRNPs