Download Biochemistry Lecture 22

Transcription
Chapter 26
Genes
• Nucleotide seq’s w/in DNA
– ~2000 genes for peptides in prokaryotes
– ~50,000 genes for peptides in eukaryotes
• DNA is not DIRECT template for peptides
– DNA = template for RNA (specifically mRNA)
– Synth mRNA from DNA = transcription
– So DNA transcribed to mRNA
• mRNA used to translate genetic code 
peptide (next lecture)
RNA Is Similar to DNA
• Both nucleic acids
• Both composed of 4 nucleotides: A, G, C
– BUT RNA has U, not T
• Both have form of ribose sugar
– BUT RNA has ribose, DNA deoxyribose
• Both linked by phosphodiester bonds
–  Sugar-phosphate backbone
– BUT RNA in single-strand (not dbl helix)
• Strand can fold back on itself
• Can form intrastrand helices, other 2o structures
Transcription DNA  RNA Similar
to Repl’n DNADNA
• Complementarity
– Base seq daughter DNA complementary to DNA
template (parent) strand
– Base seq mRNA complementary to DNA template
strand
• Initiation, Elongation, Termination Processes
• Polymerases catalyze syntheses of new nucleic
acid
– Free 3’ –OH attacks –PO4 of incoming triphosphate
– Pyrophosphate (PPi) is released
– (NMP)n + NTP  (NMP)n+1 + PPi
Transcription DNA  RNA
Similar to Repl’n DNADNA
• Template strand is read 3’  5’
– So copied strand is synth’d 5’  3’
– Complementary strands are antiparallel
• DNA double helix must be unwound in
both
– Topoisomerases impt to relieve tension on
helix in both
Transcription DNARNA Different
than Repl’n DNADNA
• Amt DNA copied
– Repl’n: entire chromosome copied
• Both strands of dbl helix copied
– Transcr’n: only 1 gene (part of
chromosome) from 1 strand of double helix
is copied
•  Single strand mRNA
– BUT gene is copied more than once
– Yields many transcripts of same gene
• Each strand of DNA being transcribed has different name
– RNA transcribed from DNA template strand (26-2)
– Complementary strand of dbl helix is called DNA nontemplate
strand or coding strand
» This strand has same seq as RNA transcript, except for
one difference
» How is it different from transcript??
Transcription Is Different – cont’d
• Origin
– Repl’n: one origin in E. coli
• What’s that called?
– Transcr’n: Enz’s/prot’s must know where along
length of DNA to begin copying/stop copying
• Polymerase
– Repl’n: DNA polymerase
• Several types w/ varied subunits
• Has proofreading ability
• Requires primer
• Elongation up to 1,000 nucleotides/sec
Transcr’n Is Different -- cont’d
• Polymerase –
cont’d
– Transcr’n: RNA
polymerase (26-4)
• 1 complex w/ 6
subunits
– Called
“holoenzyme”
– 1 subunit (s)
directs rest of
enz to site of
initiation of
transcr’n
Transcription Is Different – cont’d
– Transcr’n: RNA polymerase – cont’d
• No proofreading
• No primer needed
– Begins mRNA w/ GTP or ATP
• Elongation ~50-90 nucleotides/sec
• Unwinding
– Repl’n: helicases are used
– Transcr’n: RNA polymerase keeps ~17 bps
unwound
E. coli Promoter Region
• DNA seq @ which transcr’n
apparatus comes together to begin
copying the gene
– So each gene has a promoter
• Consensus DNA seq’s -- highly
conserved in both seq and location
(26-5)
E. coli Promoter – cont’d
• Consensus DNA seq’s – cont’d
– +1 base = first nucleotide to be transcribed
• Usually a purine
• What are the purines??
– -10 region (toward 3’ end of template strand) = 6
nucleotide seq w/ consensus TATAAT
– Spacer = ~16-18 nucleotides
– -35 region = 6 nucleotide seq w/ consensus
TTGACA
– -40  -60 region = AT-rich region = Up-stream
Promoter (UP element)
Fig.26-5
E. coli Promoter – cont’d
• When pattern met exactly
– RNA polymerase recognizes most efficiently
– Get rapid transcription
• When pattern varies from consensus
sequences
– Takes longer for RNA polymerase to
recognize promoter
– Get longer time of transcription
Initiation of Transcr’n in
E. coli (26-6)
 s subunit of RNA polymerase searches for
promoter region
– Scans ~2000 nucleotides/~ 3 sec along template
strand
• Holoenzyme binds at promoter region 
“closed complex”
– DNA bound to holoenzyme is intact
• About 15 bps unwound @ -10 region  “open
complex”
– Probably conform’l changes in polymerase enz
assist in “opening”
Initiation of Transcr’n – cont’d
• Now transcription
initiated w/
nucleotides
matched to
template strand,
added to polymer
– After ~8-9
nucleotides
added, s subunit
dissociates
• Can scan
another region
to find another
promoter
Initiation of Transcr’n – cont’d
• Regulation of transcr’n
– Strength of consensus at promoter
region, as mentioned
– Some polymerases have >1 s subunit
• Cell stress  use of diff s subunit, specific
for partic promoters needed to alleviate
specific stresses
Initiation of Transcr’n – cont’d
• Regulation of transcr’n – cont’d
– Proteins may bind DNA seq’s in/around promoter
• Some attract RNA polymerase to promoter region
– So activate transcr’n of these genes
• Some block RNA polymerase from binding @ promoter
– Called “repressors”
– So repress transcr’n of these genes
• Proteins respond to metab, repro, stress conditions w/in the
cell
– Conditions may require much peptide or depletion of peptide
– REMEMBER: Mech’s by cell to regulate glycolysis/metab??
Elongation of Transcr’n in
E. coli
• Holoenzyme free to move along
template chain
– Freer w/ dissoc’n of s subunit
– Forms “transcription bubble”
• Contains holoenzyme, template strand, new
RNA strand
Elongation of Transcr’n – cont’d
• New RNA
strand
“transiently”
base-paired to
template DNA
strand (26-1)
–  DNA-RNA
hybrid
Elongation of Transcr’n – cont’d
• DNA helix
rewinds
behind
transcription
bubble
(26-1)
Elongation of Transcr’n – cont’d
• Error rate in transcr’n ~1/105 bases added
– Much higher than in repl’n
– Acceptable
• Cell will make many transcripts of same gene
• Most  proper (active) peptides
• Some  improper peptides that can be
accommodated by cell
– What if template strand were mutated?
Termination of Transcr’n in
E. coli
• Need RNA polymerase to be processive
– If falls off, must re-start @ promoter
• What might happen in cell if problem w/ RNA
polymerase processivity?
• BUT may pause @ certain template strand
seq’s
• Some template strand seq’s cause RNA
polymerase to stop
Termination of Transcr’n – cont’d
• Two types of termination in E. coli
– Rho (r) independent (26-7)
• Template seq  RNA transcript w/ selfcomplementary nucleotides
– ~ 15-20 nucleotides
– G-C rich, followed by A-T rich regions
– Transcript forms stable hairpin loop
• Template has string of A nucleotides  string of
U nucleotides in transcript @ 3’ end
– Causes RNA polymerase to pause
Termination of Transcr’n – cont’d
– Rho (r)
independent –
cont’d
• Stable hairpin of
transcript,
followed by
relatively
unstable A-U
pairings of DNARNA hybrid 
RNA transcript
dissociates
Termination of Transcr’n – cont’d
– Rho (r) independent -- cont’d
• Polymerase dissociates
• DNA helix reanneals, rewinds
 r dependent
 r = protein = termination factor
• Binds RNA transcript @ partic binding sites
• Moves along new transcript 5’  3’ to transcr’n bubble
• Finds elongation paused
• Disrupts DNA-RNA hybrid
– Mechanism unknown
– Has ATP hydrolysis ability
Prokaryote Transcription
• Prokaryote chromosome in cytoplasm
– No organized nucleus
• Prokaryote chromosome simple
– mRNA transcr’d directly from DNA seq
– No introns/exons; “junk” DNA; etc.
• As mRNA synth’d, almost immediately
translated  peptide
EukaryoteTranscription
• More complex, less understood
• 3 RNA polymerases – I, II, III
– Each w/ specific function
– Each binds diff promoter seq
• RNA Polymerase I
– Transcribes some rRNA’s
• RNA Polymerase III
– Transcribes tRNA’s and rRNA’s
EukaryoteTranscription – cont’d
• RNA Polymerase II
– Transcribes mRNA (so most impt to
transcr’n process)
– Many subunits
– Recognizes many promoters
– Requires transcription factors
EukaryoteTranscription – cont’d
• Transcription factors (Table 26-1)
– Proteins
– Modulate binding of RNA polymerase II
to promoter region
– Complex w/ RNA polymerase  proper
binding to template, proper elongation
(26-9)
Fig.26-9
EukaryoteTranscription – cont’d
Takes place in nucleus
– mRNA transcript  cytoplasm for translation
• For peptides to be used outside the nucleus
• REMEMBER: nuclear membr has pores
• Euk genes complicated
– REMEMBER: introns/exons, “junk?” DNA
– Polymerase doesn’t seem to distinguish
• Euk DNA transcr’d directly  mRNA
• Yields a primary transcript directly reflecting entire gene
and any introns/junk
EukaryoteTranscription – cont’d
– For functioning
peptide, intron seq’s
excised before
translation
• So primary mRNA
transcripts are
spliced, rejoined
• Through
transesterification
reaction (26-13)
• Similar to
topoisomerase
mechanism
EukaryoteTranscription – cont’d
Euk genes complicated – cont’d
– Intron seq’s excised – cont’d
• Most nuclear mRNA’s spliced by specialized
RNA-protein complexes
– snRNP’s = small nuclear RiboNucleoProteins
– About 5 RNA’s + 50 prot’s complex 
spliceosome (26-16)
– Get “lariat” structure of intron seq nucleotides
– Get attack by exon –OH end  phosphate @
other exon end
Fig.26-16
EukaryoteTranscription – cont’d
– Euk mRNA’s also
further modified at
ends
• 5’ cap
– 7Methylguanosine
added @ 5’ end
– Get 5’, 5’triphosphate
linkage (26-18)
– May be impt in
initiation of
translation
EukaryoteTranscription – cont’d
– Euk mRNA’s also
further modified
at ends – cont’d
• 3’ polyA tail
– 80-250
adenylate
residues (26-19)
– May stabilize
mRNA against
enz destruction
EukaryoteTranscription – cont’d
– Final transcript = mature mRNA (26-20)
Fig.26-11
Inhibition of Transcription
by Antibiotics
• Actinomycin D (26-10)
– Planar, non-polar
– Intercalates between nucleotide bases
of DNA
• Esp. between G-C’s in G-C rich seq’s
– Now polymerase can’t move along DNA
template
Fig.26-10