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Molecular Genetics > Lecture 18+19 > Flashcards

Lecture 18+19 Flashcards

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1
Q

primary transcripts

A

most RNA moelcules are synthesized as biologically inactive precursors

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2
Q

RNA processing facts

A
  • All tRNAS are synthesized as larger pre- tRNAs
  • all large rRNAs are synthesized as a single very large precursor
  • most of small non coding RNAs are modifed
  • all mRNAs in eukaryotes must be modified
  • most bacterial mRNAs are not modified
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3
Q

types of RNA processing

A
  • cutting/cleavage, trimming, splicing
  • modification
  • editing
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4
Q

cleavage

A

cutting exons from introns

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5
Q

splicing

A

removing introns and gluing exons together

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6
Q

modification of nucleotides

A

5’ capping (m7 GPPP)
Polyadenylation (AAAA)

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7
Q

editing

A

base modification - change base (A –> G)
base insertion (C–>CC)
Base deletioon (GUG –> GG)

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8
Q

RNA processing in prokaryotes

A

pre-rRNA containing all rRNA sequences+ tRNAs is cleaved at the arrows by endo ribonucleases

individual pieces are trimmed by exonuclease

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9
Q

tRNA processing in eukaroytes

A

the precursors are cut and trimmed by appropriate ribonucleases

many bases in tRNAs are also modified

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10
Q

monocistronic (prokaryotes)

A

shine-dalgarno sequence in bacteria only
produces one protein

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11
Q

polycistronic (prokaryotes)

A

produces multiple proteins

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12
Q

All eukaryotic transcripts

A

synthesized as nonfunctional precursors (primary transcripts) and must be modified

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13
Q

RNA pol I and Pol III products

A

are cut and trimmed, and usually edited

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14
Q

RNA pol II products

A

ALWAYS modified
- G-cap is added at 5’ end DURING transcription
- introns are removed during and after transcription
- polyA tail is usually added at 3’ end after transcription
- some mRNAs are edited

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15
Q

RNA pol I product

A

45S RNA (pre rRNA), is processed to 5.8s, 18s, and 28s in nucleolus with an assist of snoRNAs (small nuclear RNAs)

mnay bases are modified by methylation
the transcrupt is cleaved and trimmed

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16
Q

tRNA procesing in eukaryotes

A
  • both the 5’ and 3’ ends are trimmed (in all tRNAs) by RNAse P and D respectively
  • CCA added to the 3’ end
  • introns (when present) are removed by specialized endonulcease/ligase
  • some bases are modified (in all tRNAs) to provide stability and enhance the functioning of the mature tRNA
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17
Q

capping

A

5’ ends of nascent transcripts

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18
Q

splicing

A

removal of introns

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19
Q

cleavage

A

3’ ends are generated by cleavage. NOT termination

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20
Q

CTD of RNAP II coordinates

A
  • capping
  • splicing
  • cleavage
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21
Q

promoter escape

A

capping

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22
Q

elongation

A

splicing

23
Q

eukaryotic mRNAs are capped at 5’ end

A
  • protects 5’ end from exonuclrases
  • necessary for translation
  • 5’ cap: residue of 7 methylguanosine (7-MeG)
  • 7-MeG is attached to the 5’ end of terminal residue of EVERY RNA via 5’-5’ - triphosphate linkage
  • caps may differ by methylation pattern
24
Q

1st modification state

A

(phosphorylation by TFIIH) of CTD allows promoter clearance and recruitment of “capping enzyme” –> Guanylyltranspherase

  • the 5’ cap is formed by condensation of a molecule of GTP with triphosphate at the 5’end of the transcript (5’-5’ - Triphosphate linkage)

The guanine is subsequently methylated at N -7 and additional methyl groups are added to the 2’ hydroxyls of the first and second nucleotides adjacent to the cap

25
Q

5’ cap synthesis

A
  • Guanylyltransferase (the capping enzyme) is associated with the Pol II CTD to ensure that each mRNA is capped as it is transcribed
  • Once the cap is complete, guanylyltransferase dissociates and the cap-binding complex CBC binds.
26
Q

Termination of transcription RNA pol II

A
  • Pol II termination does not occur at a conserved site or at a constant distance from the 3’ end of mature RNAs
  • Mammals: take place anywhere from a few kb to several kb pairs downstream from the 3’ end of the mature transcript
  • ## Polyadenylation signal (AAUAAA) is present in primary transcript. And is directly encoded by the DNAFactors responsible for cleavage: of the primary transcript bind to the AAUAAA sequence, resulting in cleavage somewhat downstream from that position.
27
Q

CPSF

A

cleavage and polyadenylation specific factor

28
Q

CstF

A

cleavage stimulation factor

29
Q

generating 3’ end of eukaryotic mRNA

A

CPSF and CstF are recruited to PolyA signal sequence (AAUAA) from CTD

Endonuclease cuts just upstrewam from GU-rich sequence

30
Q

addition of the 3’ poly A tail to the transcript

A
  • the 3’ poly A tail typically 80-250 A residues
  • serves as a binding site for one or more specific proteins that help protect mRNA from enzymatic destruction
  • PAP (poly A polymerase) adds a stretch of A’s to the 3’ generting a poly A tail - POST TRANSCRIPTIONALLY
  • Poly A binding (PABPs) bind to Poly A tail - protecting it from 3’ to 5’ exonucleases
31
Q

RNA splicing

A

all pre mRNA splicing mechansisms consist of the ordered breaking and joining of specific phosphodiester bonds to achieve the precise excesion of introns

Splicing must be carried out quickly and correctly to produce the mRNAs required for protein production

32
Q

Duchenne muscular dystrophy and cystic fibrosis

A

caused by aberrant pre-mRNA splicing

33
Q

splice junctions

A
  • sequences within RNA determine where splicing occurs
34
Q

5’ splice site is always

A

GU

35
Q

3’ splice site

A

AG

36
Q

generic intron would have seuqence

A

GU……AG

minority of introns have AU….AC

37
Q

accurate and efficient splicing relies on

A

base pairing between the pre-mRNA and the splicing machinery to specify the bonds to be broken or formed.

38
Q

branch point (splice junctions)

A

approx 20-50nt upstream from 3’ splice site

the surrounding sequences and perhaps the structure of the pre-mRNA itself must play a role in the selection of splice sites.

accurate and efficient splicing relies on base pairing between pre-mRNA and the splicing machinery to specify the bonds to be broken or formed

39
Q

the splice reaction

A
  1. two site speciic transesterification reactions
    - resulting in phosphodiester bond cleavage and ligation
  2. formation of lariat (lasso, loop and tail structure)
  3. introns released and exons are joined together
  4. splicing requirements: splice sites and the branch point site

these reactions are catalyzed by ribonucleoproteins (RNPs)

40
Q

RNPs

A

complexes of non-protein coding RNAs and proteins

41
Q

spliceosome - catalyzes most pre-mRNA splicing

A

large complex of 5 small nuclear ribonucleproteins (snRNPs snrups) + hundreds of additional protein components

-

42
Q

snRNAs

A

single small nuclear RNAs (100-300nt) = snRNAs (U1,U2,U4-6) - in each snurp

Snurps have a name corresponding to snRNA

Each snRNA is complexed with several (under 20) proteins forming snRNPs (snurps)

43
Q

the spliceosome

A

snRNPs (with help from proteins have several functions)
- recognition of 5’ and 3’ splice sites, and the branch site bringing those sites together; the catalysis of cleavage and joining reactions

44
Q

splicing relies on many specific intermolecular reactions

A

protein - protein
protein - rna
rna - rna

45
Q

correct splicing relies on

A

sequential assembly and rearrangement of spliceosome on the intron to be removed

base pairing between the snRNAs of the spliceosome and the pre-mRNA allows cells to select correct splice sites.

46
Q

splicing process

A
  1. U1 binds to the 5’ splice site; U2 binds to the branch point
  2. U4-U5-U6 trimeric snRNP displaces U1 at the 5’ splice site, then U4 dissociates.
  3. U6 and U2 catalyze attack of the branch point on the 5’ splice site
  4. the 5’ splice site attacks the 3’ splice site, completing the reaction
47
Q

self splicing

A
  • transcripts other than nuclear pre-mRNA may contain introns that will be spliced
  • all non nulcear transcripts do not use spliceosome
  • they are self-spliced and the catalysis is performed by the intron itself (ribozyme) No involvement of any protein enzymes
  • ## these transcripts belong to two classes: group I and group II based on the mechanism of the 1st transesterification reaction
48
Q

nuclear pre-mRNA

A

abundance: very common, used for most eukaryotic genes

mechanism: two transesterification reactions; branch site A

major and minor splicosomes

49
Q

group II introns

A

abundance: rare; some eukaryotic genes from organelles and prokaryotes

mechanism: same as pre mRNA

RNA enzyme encoded by intron (ribozyme)

50
Q

group I introns

A

abundance: rare; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genes

mechanism: two transesterification reactions; requires a guanine nucleoside or nucleotide cofactor (not used as a source of energy) for the firdt transesterification

51
Q

group I intron mechanism

A

the 3-OH of guanosoine acts as a nucleophile, attacking the phosphate at the 5’ splice site

the 3-OH of the 5’ exon becomes the nucleophile, completing the reaction

52
Q

group II introns mechanism

A

the structure of the RNA itself, rather than the assembly of multiple snRNPs, creates an active site for catalysis

53
Q

splicing mechanisms and requirements

A
  • group I nad II introns self splice
  • the information for splicing including catalytic activity is present in group I and II introns
  • all 3 classes of splicing reactions proceed by two transesterification
  • most of the self splicing intron sequence is critical

Molecular Genetics (9 decks)

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