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 DNADNA

• 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 DNADNA

• 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 DNARNA Different

than Repl’n DNADNA• 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 () 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) 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, 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 subunit

• Cell stress use of diff 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 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 () independent (26-7)

• Template seq RNA transcript w/ self-complementary 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 () independent – cont’d

• Stable hairpin of transcript, followed by relatively unstable A-U pairings of DNA-RNA hybrid RNA transcript dissociates

Termination of Transcr’n – cont’d

– Rho () independent -- cont’d

• Polymerase dissociates

• DNA helix reanneals, rewinds

dependent

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

– 7-Methylguanosine 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