Chapter 29 Rna Synthesis And Processing

Front 1

most abundant RNA

Back 1

ribosomal RNA

Front 2

RNA polymerase

Back 2

enzyme catalyzing RNA transcription
template is anti-sense strand of DNA
requires Mg (interact with Asp residues)

Front 3

structure of prokaryotic RNA polymerase

Back 3

2 alpha subunits, beta and beta' subunits
2 functional states: holoenzyme contains those + sigma for initiation
after initiation, sigma is released--now is core enzyme for elongation and termination

Front 4

identifying promoter sequence in prokaryotic RNA transcription

Back 4

search for intiation site--consensus sequence TTGACA (or something like that) at -35, TATAAT at -10 (or something like that); sigma subunit recognizes this

Front 5

unwinding of DNA double helix in prokaryotic RNA transcription

Back 5

unwinds ~17bp (becomes open promoter complex)
first nucleotide is either ATP or GTP

Front 6

elongation of prokaryotic RNA transcription

Back 6

goes from holoenzyme to core enzyme, which binds more strongly to DNA
no repair function (no exonuclease activity)
"transcription bubble"--region containing DNA, RNA, RNA polymerase

Front 7

rho-independent stop signal for prokaryotes

Back 7

there is a sequence with a palindromic GC-rich region that will form stem loop (pauses RNA polymerase); followed by AT-rich region--these interactions are very weak, so RNA dissociate

Front 8

rho-dependent transcription termination

Back 8

rha binds ~72 bases in single-stranded RNA
ATP-dependent helicase
particular RNA sequence activates ATPase of rho protein and this pulls RNA out of transcription complex

Front 9

RNA modification in prokaryotes
(+ 3 kinds of modification)

Back 9

mRNA: little modification
tRNA & RNA: extensive modifications
1) cleavage (ex. ribosomal and transfer RNA precursor separated to become mature RNA)
2) addition of nucleotides to RNA termini (ex. post transcriptional CCA addition to tRNA)
3) base modification (ex. methylation, modified uridylate)

Front 10

rifampicin

Back 10

antibiotic used to treat TB
blocks channel into which RNA-DNA hybrid must pass
inhibits initiation/elongation of RNA transcript
rifampicin-binding pocket is not conserved in eukaryotes, so can't harm eukaryotic cells

Front 11

differences between eukaryotic and prokaryotic RNA transcription

Back 11

-prokaryotic transcription and translation can happen simultaneously, while eukaryotic transcription and translation are temporally and spatially distinct (mRNA must be transported out of nucleus to cytoplasm)
-more complex transcriptional regulation for eukaryotes (uses transcription factors)
-mRNA processing--eukaryotic mRNA is extensively modified

Front 12

type I eukaryotic RNA polymerase

Back 12

exists in nucleolus
transcribes 18S, 5.8S, 28S rRNA
insensitive to alpha-amanitin
several hundred tandem repeats on gene, so lots of rRNA

Front 13

type II eukaryotic RNA polymerase

Back 13

exists in nucleoplasm
transcribes mRNA and snRNA
strongly inhibited by alpha-amanitin

Front 14

type III eukaryotic RNA polymerase

Back 14

exists in neucleoplasm
transcribes tRNA and 5S rRNA
inhibited by high concentrations of alpha-amanitin
promoter sites is within transcribed gene

Front 15

modification of transcription products of RNA pol I

Back 15

RNA polymerase I
18S, 5.8S, and 28S are originally all one sequence
nucleotide modification occurs (addition of methyl groups and pseudouridine) then cleavage to mature rRNAs

Front 16

tRNA modification in eukaryotes

Back 16

-cleavage
-intron splicing
-addition of nucleotides--CCA added to 3' end
-base modification

Front 17

RNA polymerase II promoters

Back 17

enhancer-----//----TATA box---Inr----
or
enhancer----//----Inr------DPE----
Inr=initiatior element
DPE=downstream promoter element
transcription factors recognize promoter site and recruit RNA polymerase to initiate

Front 18

enhancers in RNA polymerase II promoters

Back 18

no promoter activity, but helps transcription from promoter site
can be found anywhere (upstream, downstream, etc)

Front 19

transcription initiation by RNA polymerase II

Back 19

TATA-box binding protein (component of TFII D) binds to TATA box
recruits TFII A, B, F, RNA pol II, TFII E, and H
TFII H has helicase and kinase activity, so opens up DNA and phosphorylate C terminal domain of RNA polymerase II--this allows RNA polymerase to leave from complex and start elongation

Front 20

eukaryotic RNA transcription termination

Back 20

no strong termination sequence
3' end of mRNA is cleaved--AAUAAA is recognized by endonuclease which cuts between U and A

Front 21

capping (RNA processing)

Back 21

5' end of mRNA is capped (tRNA and rRNA are not capped)
GTP binds to first nucleotide of mRNA (5'-5' triphosphate linkage)
terminal G is then methylated
required for further processing of mRNA transcript
protects mRNA from degradation by phosphatases and 5' exonucleases
enhances translation

Front 22

poly(A) addition

Back 22

most mRNAs get poly-adenylate addition at 3' end
enzyme: poly(A) polymerase
post-trancriptional modification (not coded by DNA)
don't know role of poly(A) tail, but may increase stability--some mRNAs don't have poly(A) tail (i.e. histones)

Front 23

RNA editing in eukaryotes

Back 23

modification of nucleotides after transcription by processes other than splicing--generates molecular diversity
ex: apolipoprotein B-100
in unedited form there is CAA in middle--in liver, this is translated as is
in small intestine during development, C can be deaminated to U, so now UAA is a stop codon resulting in a short protein--APO B-48

Front 24

splicing in eukaryotic RNA

Back 24

genes are composed of exons and introns--introns are spliced out
splice site consensus sequence:
AG/GUAAGU---------A--------CAG/G
GU A AG= invariant--these sequences are recognized by splicing enzymes

Front 25

splicing mechanism

Back 25

no energy required--transesterifications
2' OH group of A attacks phosphoryl of G, so releases exon leaving lariat intermediate
3' OH of exon attacks phosphoryl of G next to other exon so this releases forming spliced product
lariat form of intron is left

Front 26

snRNAs

Back 26

catalyze splicing
contain fewer than 300 nucleotides
mechanism: U2 and U2 join by base pairing and recruit U4, U5, and U6
U6 is enzyme, so U1 and U4 are cleaved by U2
catalyze first transesterification then second transesterification leaving lariat intron and spliced exons

Front 27

spliceosomes

Back 27

large complexes of snRNPs, hundreds of proteins called splicing factors and precursor mRNAs

Front 28

small nuclear ribonucleoproteins

Back 28

snRNAs and associated proteins

Front 29

alternative splicing

Back 29

different combinations of exons from the same gene can be spliced into a mature RNA
allows one gene to encode for more than one protein
i.e. a certain RNA strand becomes CGRP in neuronal cells and calcitonin in thyroid cells
splicing site selection is determined by splicing factors

Front 30

hemoglobin beta chain gene and defective splicing

Back 30

normal pre-mRNA has stop codon in intron
pre-mRNA with mutation in intron makes splice site after stop codon, so abnormal mature RNA has a stop codon, so makes shorter proteins which are degraded
causes thalassemia