1. DNA contains the genetic code and is replicated and transcribed into mRNA, which is then translated into protein. During replication, DNA polymerase adds nucleotides to the growing DNA strand while helicase unwinds the double helix. 2. Transcription involves RNA polymerase binding to DNA and synthesizing mRNA, which then undergoes processing. Translation uses tRNA to decode the mRNA codon by codon, adding the corresponding amino acids specified by the genetic code to form a polypeptide chain. 3. Both transcription and translation are complex processes involving many proteins and enzymes to proofread and maintain fidelity. DNA, RNA and proteins are synthesized through the coordinated actions of replication, transcription and translation.

1. Chapter 2
Macromolecular Synthesis and
Processing: DNA, RNA, and
protein Synthesis

2. DNA
• Disruption of this molecule can have a ripple
effect on potentially hundreds of biochemical
processes.
• DNADNAmRNAProtein
• Replicationtranscriptiontranslation

3. Polarity
• Antiparallel
• One structure begins with
a 3’OH and ends with a
5’PO4

4. 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

5. 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

6. 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

7. DNA gyrase
• Tetrameric enzyme
• Relieves strain while
DNA is unwound by
helicase.

8. 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

9. DNA Replication
DNA 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

11. 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

13. E. coli DNA replication

14. 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.
– XerCholliday
junctionXerD/FtsK
• Partitioning of the two chromosomes
into two separate daughter cells with
the aid of FtsK.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.

21. 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

22. 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.

23. 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

24. 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)

25. 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).

27. 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

28. 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

29. 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

30. Translation
• mRNAprotein
• 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

31. 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.

32. tRNA

33. • 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

34. Aminoacyl-tRNA synthetase

35. Ribosome