Ch 13: The Genetic Code and Transcription

Central Dogma

DNA —transcription—> RNA —translation—> protein

RNA polymerase

directs synthesis of RNA using a DNA template

• 5’ —> 3’ direction

• no primer required

• enzyme uses NTPs

RNA synthesis

mRNA

intermediate molecule between DNA and proteins, carries info for order of amino acids

n(NTP) + DNA tempalte —RNAP—> (NMP)n n(PP_i)

reaction of RNA synthesis

• best understood so underlies all we know

• many features conserved

• target of antibiotics

Why start with prokaryotes?

1. Initiation

2. Elongation

3. Termination

Transcription stages

holoenzyme

whole enzyme

σ

RNAP core enzyme lacks…

• only 1 RNAP that produces all types of RNA

• mRNA not modified after synthesis

• genes arranged in polycistronic operons

• transcription and translation coupled

Prokaryotic transcription

interacts with DNA and transcription factors to play regulatory role

α E.coli polymerase subunit

nonessential and regulatory

ω E.coli polymerase subunit

bind DNA and synthesize RNA

β and β’ E.coli polymerase subunit

• promoter recognition during initiation

• not present during elongation

• confer specificity allowing different genes to be expressed

σ E.coli polymerase subunit importance

-35 = TTGACA and -10 = TATAAT

E.colli promoter sequences

consensus sequence

a sequence of DNA nucleotides most frequently found at each position and is necessary for particular function

σ₇₀

Major E.coli σ factor

promoter

where RNAP binds to start transcription, upstream of sequences

open-complex

part of initiation where single-stranded bubble created to go from stable, closed complex to active site

• RNAP finds and recognized promoter via σ subunit and -35 and -10 sequences

• RNAP binds and partially unwinds DNA creating open complex formation

E.coli initiation steps

Promoter regions of many genes were aligned by start site of transcription and found to have similar sequences at set distances

How were consensus sequences discovered?

40 nt/second

RNAP rate (nt/sec)

800 nt/sec

DNAP III rate (nt/sec)

15 aa/sec moving at 45 nt/sec

Prokaryote ribosome rate (aa/sec and nt/sec)

template strand (nonsense strand)

being copied, anti-parallel to RNA

coding/sense strand (makes “sense” when translated)

looks like mRNA

• RNAP moves away from promoter with core enzyme synthesizing RNA in 5’ —> 3’ direction

  • 3’-OH = nucleophile to attack α phosphate on incoming nucleotide

• σ releases

E.coli Elongation steps

Rho independent and Rho dependent

E.coli termination types

• no protein required, based on nucleotide sequence

• RNA folds into hairpin with G-C rich stem followed by string of U

• rU-rA bonds stronger than rU-dA —> weakest interaction left with DNA causing dissociation

Rho-independent termination

• hexamer that recognizes rut base sequence

• wraps RNA around itself

• uses ATP to pull RNA from DNA

Rho-dependent termination

operon

a cluster of related genes under control of a single promoter

• multiple RNAPs

• occurs in nucleus

• not coupled with translation

• transcription occurs on chromatin

  • requires chromatin remodeling

• require processing to produce mature mRNAs

Differences in eukaryotic transcription vs prokaryotic transcription

rRNA

RNA Pol I products

mRNA, snRNA

RNA Pol II products

tRNA, 5S rRNA

RNA Pol III products

12 subunits (10 required)

RNA Pol II subunits

• Which cells gene expressed

• When gene expressed

• How much gene expressed

What do enhancers and repressors regulate?

core promoter elements

required for direct accurate initiation of transcription by RNA Pol II machinery

• recognizes promoter and assembles initiation complex

• recruits other GTFs and RNA Pol II

General transcription factors (GTPs) roles

• GTF (TF_IID) recognize promoter sequence and bind

• Recruit other GTFs and RNA Pol II

• Pre-initiation complex formed

Eukaryote Initiation step

• GTFs phosphorylate CTD

• machinery disassembles and RNA Pol II leaves

• RNA Pol II adds first NTP without 3’-OH

• Bridge helix moves DNA and RNA during elongation

• mRNA processing

Eukaryote Elongation steps

hnRNA (heterogenous nuclear RNA)

initial mRNA name

• unusual 5’-5’ triphosphate linkage

• binds to phosphorylated CTD and moves to mRNA

• recognized by translation factors and protects against exonucleases

Eukaryotic 5’ 7-methyl-G-cap

introns

regions of the initial RNA transcript that are not found in the mature mRNA

U1 snRNP

spliceosome example

1. U1 snRNA binds to 5’ splice site of intron via RNA-RNA interaction

2. U2 and hnRNA branch point (A) bind

3. U1 and U2 interact

4. other SnRNPs come in

5. 2’-OH on 3’ branch site attacks phosphodiester bond art 5’ splice junction

6. 3’-OH generated and Lariat loop forms

7. 3’-OH attacks 3’ splice site

8. Mature mRNA created and Lariat intron degraded

Mechanism of spliceosome splicing

1. GTP binds to active site in intron

2. 3’-OH on GTP breaks phosphodiester bond at 3’ left exon

3. New 3’-OH interacts with phosphodiester bond on right exon

4. intron sliced out and exon joined

Self splicing mechanism

1. CPF recognizes sequence in mRNA

2. CPF cuts mRNA and adds polyA tail

3. Ral1/Rat1 RNase degrades 5’ end moving towards RNA Pol II causing dissociation from DNA

Eukaryote Termination

guide RNAs (gRNAs)

What directs post-transcriptional modifications

changing base or insertion/deletion

What edits can be made post-transcriptionally

TATA box

A short nucleotide sequence 20–30 bp upstream from the initiation site of eukaryotic genes to which RNA polymerase II binds

multiple codons per amino acid

What does it mean that the genetic code is degenerate

each codon is for only one amino acid

What does it mean that the genetic is unambiguous

via degeneracy and chemically similar aa’s sharing similar bases in codon

how does the genetic code buffer against mutations?

AUG

start codon

UAG, UAA, UGA

stop codons

methionine

What does AUG code for?

formylated-methionine (fMet)

Bacteria starting amino acid

wobble hypothesis

hydrogen bonding between the codon and anticodon at the 3rd position is subject to modified base-pairing rules