Chapter 2

Macromolecular Synthesis and Processing: DNA, RNA, and

protein Synthesis

DNA

• Disruption of this molecule can have a ripple effect on potentially hundreds of biochemical processes.

• DNADNAmRNAProtein• Replicationtranscriptiontranslation

Polarity

• Antiparallel• One structure begins with

a 3’OH and ends with a 5’PO4

Major and Minor groove• Usually exists in a stable B conformation.• A conformation (11 base-pairs instead of 10 base-pairs per turn)• Z-DNA is left-handed rather than right-handed helix having 12

base-pairs per turn.• Most often formed during extended runs of G-C

B A Z

E. coli chromosome

• 1.5 mm long• In an actively growing cell, DNA comprises 3-4% of

the cellular dry mass.• There are about 3 genomes in an actively growing

cell.• Topoisomerases are enzymes that

alter the topological form (supercoiling) of the circular DNA molecule

Topoisomerases• Two types (I and II) are further subdivided…• IA nicks one strand of DNA and passes the other strand through

the break relaxing the negatively supercoiled DNA.• IB (Top III E. coli) which relaxes the negatively supercoiled DNA by

breaking of one of the phosphodiester bonds in dsDNA, allowing controlled rotational diffusion (“swivel”) of the 3’-OH end around the 5’-P end.

• Both IA and IB subtypes reseal the nicked phosphodiester backbone.

• Type II requires energy to underwind DNA and introduce negative supercoils

http://www.youtube.com/watch?v=EYGrElVyHnU

DNA gyrase

• Tetrameric enzyme• Relieves strain while

DNA is unwound by helicase.

Nucleoid domain• A cell 1um in length must accommodate a

chromosome 1500um in length.• It must do this in a way that does not tangle the

molecule!!!!• The bacterial chromosome actually contains

between 30 and 200 negatively supercoiled loops or domains

• Each domain represents a separate topological unit, the boundaries defined by sites on the DNA and bound to anchor proteins

• Gathering the DNA loops at their bases would compact the chromosome to the radius of 1um.

• 4 different histone-like molecules contribute to this “compactasome”.– HU, H-NS, Fis, AND RNApol

DNA ReplicationDNA replication begins with a short

primer—a starter strand.

The RNA primer is complementary to the DNA template.

Primase—dnaG locus an enzyme—synthesizes a short RNA primer elongated by DNA polII one nucleotide at a time.

Rnase H– an RNA degrading enzyme that recognizes the RNA : DNA hybrid that erases these RNA primers

DNA polymerase adds nucleotides to the 3′ end.

DNA helicase uses energy from ATP hydrolysis to unwind the DNA.

Single-strand binding proteins keep the strands from getting back together. http://www.youtube.com/watch?v=up-ewMIsxy0

How fast does it go?

• 4,639,221 bp• 800-1000 bp/second• Frequency of error is 1/1010 bp replicated

– High degree of fidelity

• Once initiated, replication is an elaborate process requiring a large number of proteins… Table 2-3

E. coli DNA replication

Termination of DNA Replication• Two daughter chromosomes form a

linked concatamer due to the topological constraints when separating the strands of a double stranded helical circle.

• Must be resolved through recombination if the cell is to divide.

• XerC and XerD are tyrosine family site-specific recombinases bind cooperatively with the 33 bp chromosomal site dif located at the replication terminus.– XerCholliday

junctionXerD/FtsK• Partitioning of the two chromosomes

into two separate daughter cells with the aid of FtsK.

RNA synthesis: transcription

• The process by which the information contained within genes is converted to RNA.

• The process is the same whether the gene encodes for mRNA, tRNA, or rRNA

• Requires a DNA-dependant RNA polymerase and proceeds in a manner similar to DNA synthesis

• Uses ribonucleic acid triphosphates (rNTPs) rather than dNTPs.

RNA Polymerase• RNAP is an extremely complex machine that senses signals

coming from numerous regulatory proteins as well as signals encoded in the DNA sequence.

• Consists of four polypeptides: αββ'σ• Core polymerase consists of 2α subunits plus one β and one

β‘ subunit.• Can bind to DNA at random sites and synthesize random

lengths of RNA.• Holoenzyme = core + σ subunit binds to DNA at SPECIFIC

sites called PROMOTERS and transcribe specific lengths of RNA.

σ70

• 70 kDa molecular weight• Considered the housekeeping σ, but there are

several specialty σ factors that direct RNAP to specific promoters.

• Plays an important role in promoter recognition by RNA polymerase.

α2ββ'σ

• β subunit carries the catalytic site of RNA synthesis as well as the binding site for substrates and products.

• β‘ subunit plays a role in DNA template binding while the two α subunits assemble the two larger subunits into core enzymes α2ββ'σ.

RNAP movement

• Upstream and downstream are to describe regions relative to the direction of RNA polymerase movement.

• The promoter is upstream from the structural gene.

• Moves in the 3’5’ direction on the DNA template strand while synthesizing RNA in the 5’3’ direction.

Transcription

• Three main steps: initiation, elongation, an termination

• Initiation: binding of polymerase to promoter with the formation of a stable RNAP-DNA initiation complex and catalysis of the first 3’5’ internucleotide bond

• Elongation: the translocation of RNAP along the DNA template

• Termination: dissociation of the complex

Initiation• DNA sequence at the promoter includes two conserved

sequences.• -10 (Pribnow’s box) and –35 (recognition site) region

are the transcriptional start points and σ factors recognize both.

Alternate σ factors

• σ -70 plays an important role in normal transcription initiation.

• Alternate σ factors can change the promoter recognition specificity of core enzyme:– σ -32 initiation heat shock response– σ -28 flagellar genes, chemotaxis– σ -24 extreme heat shock– σ -42/S stationary phase– σ -54 Nitrogen

Elongation• After 8-9 nucleotides, σ is released… decreased

affinity for the ternary complex (RNAP—DNA—nacent RNA complex)

• Transcription proceeds at a rate of 30-60 nucleotides per second.

• Rate of elongation is not uniform and there are regions where elongation is very slow called pausing sites.– G-C rich– Hairpin loop formation in RNA (RNA-RNA stem loop)

Termination

• Cessation of elongation• Release of transcript from the ternary

complex• Dissociation of polymerase from the template

– ρ-independent (G-C rich stem-loop structure) and ρ-dependent (requires the ρ-gene product causing a strong pause site a specific distance from the promoter).

RNA• Stable and unstable RNAs:

• Stable RNA is rRNA and tRNA• Unstable RNA is mRNA• In E. coli, 70-80% of all RNA is rRNA, 15-25% is tRNA, and 3-

5% is mRNA• Factors that contribute to stability:

• Secondary structure (tRNA)• Ribonucleoprotein complex (rRNA)

• Average mRNA has a half-life (time required to reduce mRNA by half) of approximately 40 seconds at 37°C.

• Degradosome complex: polynucleotide phosphorylase, Rnase E, ATP-dependent RNA helicase, DnaK chaparone, and enolase

Poly(A) tails

• Once thought to be unique to only eukaryotic mRNA, but now shown to occur in bacterial mRNA.

• Two poly(A) polymerases have been identified in E. coli: Pap I (80% of poly(A)s and polynucleotide phosphorylase (Pnp) accounts for the remainder

RNA processing• All stable RNAs and a few mRNAs of E. coli must be processed

prior to their use.– Each of the seven rRNA transcription units is trancribed in a single

message:• 5’leader-16s rRNA-spacer-23s RNA-5s rRNS-trailer-3’

– The spacer always contains some tRNA gene– Four basic types of processing:

• Precise separation of polycistronic transcripts into moncistronic precursor tRNAs

• Removal of extraneous nucleotides from 5’ amd 3’ ends• Addition of terminal residues to RNAs lacking them• Appropriate modification of base or ribose moieties of nucleosides in RNA

chain

Translation

• mRNAprotein• A codon codes for an Amino acid:– 20 naturally occurring aa– Triplet code : 64 possible combination of AGCU bases– Usually more than one triplet code that codes for a particular aa; the

code is degenerate– Nonoverlapping: the triplet code codes for a particular aa

• Nonsense codons: serve as termination signals or STOP codons

• If a single base is added or deleted, it will lead to a frameshift where the entire sequence of triplets is altered from that point forward

N-FORMYLMETHIONINE

• The initiating methionine (AUG) in prokaryotic protein synthesis• It is a derivative of the amino acid methionine in which a formyl

group has been added to the amino group. • It is specifically used for initiation of protein synthesis, and may

be removed after.• fMet plays a crucial part in the protein synthesis of bacteria,

mitichondria and chloroplasts (endosymbiosis theory). It is not used in cytosolic protein synthesis of eukaryotes and is not used by Archaea.

• In the human body, fMet is recognized by the immune system as foreign material and stimulates the body to fight against potential infection.

tRNA

• Anticodon: the triplet of bases on the tRNA that recognize the code in mRNA.

• Codon recognition site: the site that recognize the specific tRNA synthetase (ligase) that add the amino acid to the tRNA (ligase recognition site)

• Amino acid attachment site: CCA-3’• Aminoacyl-tRNA synthetase: the enzyme that specific for

each amino acid charging enzyme

Aminoacyl-tRNA synthetase

Ribosome