Assembly of procaryotic cell is about 10-fold faster than assembly of eucaryotic cell. Polymerzation of macromolecules somewhat faster, but not 10-fold. What are the requirements?

Problem is that of synthesizing 1 gm of E. coli, or about 1012 cells.

55% protein; 20.5 % RNA; 3 % DNA; 9% phospholipids; 2.5% murein (PG); 3.4% lipopolysaccharides; and 2.5% glycogen.

Semiconservative replication of DNA was postulated by Watson and Crick in 1953 paper on structure of DNA. Biochemically complex process required to complete this very simple model, proven to be correct.

All known DNA polymerase activities polymerize DNA in 5' to 3' direction, since free 3'-OH group required for nucleophilic attack on the alpha phosphate of the dNTP. Kornberg received Nobel prize for discovery of first DNA polymerase activity and for defining its activity requirements (DNA template, dNTPs, and primer with 3'-OH).

All bacterial chromosomes are circular DNA molecules (there can be more than one type of circle per cell to encode all genes).

Replication initiates an a unique ORIGIN OF REPLICATION, and ends at a TERMINUS about 180° from the origin.

Once initiated, replication proceeds at constant rate (800-1000 NT per second) in both directions from the origin (BIDIRECTIONAL REPLICATION) until the two forks reach the TERMINUS; for the entire cellular process, only 1 error is made in 1010 nt synthesized. Since rate is constant, replication is regulated by the rate at which replication initiation occurs.

DNA synthesis always proceeds in a 5' to 3' direction, since addition of base requires free 3'-OH group. Because of this fact, only one strand can be synthesized in a continuous fashion--the LEADING STRAND. The LAGGING STRAND is synthesized in segments, known as OKAZAKI FRAGMENTS.

The separation of the strands of double-stranded DNA for replication requires energy in the form of ATP.DnaB (DNA HELICASE) unwinds the DNA strands to ss-DNA; this protein moves in 5' to 3' direction on the lagging strand (see Fig. 8, p. 78). Single-stranded DNA binding protein (SSB) binds to separated strands and prevents their reassociation.

E. coli contains 3 DNA polymerases: denoted I, II, and III. Polymerases I and II are repair enzymes, although Pol I also plays a role in replication. The main replication enzyme is DNA POLYMERASE III.

Haemophilis influenzae, Mycoplasma genitalium, and Synechocystis sp. PCC 6803: Only Pol I and Pol III

Archaea: Only one recognizable DNA polymerase gene was detected in the Methanococcus jannaschii genome project. Is there only one DNA polymerase in these organisms, or is there another, very different type of DNA polymerase?

DNA synthesis requires: DNA template (single-stranded, or gapped DNA molecule)

Primer with free 3'-OH group

dNTPs (deoxynucleotide triphosphates)

DNA-dependent DNA polymerase

During synthesis, pyrophosphate (PPi) is released as 3'OH attackes the alpha-phosphate group of the incoming dNTP.

LEADING STRAND synthesis is straight-forward. DNA Polymerase III loads and proceeds to synthesize DNA in 5' to 3' direction. LAGGING STRAND is more complex, since synthesis cannot be continuous.

Direct evidence of discontinuous synthesis on LAGGING STRAND was obtained by R. Okazaki by pulse-labeling with 3H-thymidine. Much of newly synthesized DNA could be isolated as short 500-2000 bp fragments. Each fragment requires a primer, and all fragments ultimately must be joined to complete the replication process. Primers for Okazaki fragment synthesis are RNA primers synthesized by PRIMASE (DnaG) DNA polymerase I hydrolyzes the RNA primers and fills gaps with DNA. DNA ligase joins one DNA fragment to the next.

RNA polymerase doesn't need primer, so why does DNA polymerase? Intrinsic error rate of the polymerase is about 10-5. 3' to 5' exonuclease activity of Pol III allows EDITING, removal of mismatched bases prior to elongation. A truly self-correcting enzyme cannot start synthesis without a primer.

DNA Polymerase III appears to have a dimeric structure (heterodimeric (?) since the activities on the two strands is different), and movement of replication fork complex is complex; binding and release of Pol III is very slow, yet Okazaki fragment synthesis is rapid. This is a paradox --how solved???

Looping of the lagging strand occurs so that both leading and lagging strand synthesis occur at the REPLISOME structure

1. Strand separation by helicase (DnaB or Rep) and stabilization by SSB (single-stranded DNA binding protein

2. Unwinding by helicase causes positive supercoiling; DNA gyrase compensates with negative supercoiling to prevent knotting of DNA

3. Prepriming/Primosome assembly occurs at origin of replication (oriC)

4. Primase (DnaG) forms RNA primers (6-10 nt long); DNA polymerase III initiates synthesis on leading and lagging strands

5. On lagging strand, Pol III synthesizes Okazaki fragment until it reaches another RNA primer. DNA polymerase I uses its 5' to 3' exonuclease activity to remove the RNA primer and its 5' to 3' DNA polymerase activity replaces the RNA with DNA.

6. The single missing phosodiester linkage that remains is sealed through the action of DNA ligase.

E. coli has 4700 kbp of DNA; if replication rate is 1 kbp per second, it will take 2350 seconds since replication is bidirectional (about 39 minutes) for replication to occur. Fastest doubling times are about 18-20 minutes, or 2 times faster than this. Critical factor is how often replication initiates--initiation (and termination) must occur at the same rate or faster than cell division. Multiple replication forks (initiated at about 18-minute intervals) must be formed for division to occur so rapidly.

Replication in E. coli begins at a unique origin of replication (oriC) that occurs at 83.5 minutes on the E. coli genetic map. The origin is a unusual A+T rich region of the DNA, about 250 nt in length. "Artificial minichromo-somes" can be formed by cloning oriC along with foreign DNA.

Origin is highly methylated, and the origin can attach to membrane (outer membrane?). Origin contains several promoters for RNA polymerase binding, and "initiator RNA" synthesis is required for replication initiation.

There are 3 models for how initiation could be controlled, and both suggest at a certain cell mass : chromosome ratio must be attained before initiation will occur. Control could be positive or negative or some combination of both.

Positive: concentration of some element increases until critical treshold concentration is reached. When this occurs, replication begins.

Negative: at the time of initiation or shortly thereafter, inhibitor of initiation is produced that must be diluted. When dilution is sufficient, replication is initiated.

Although probably elements of both positive and negative control, mostly positive seems to exist. The critical protein is DnaA, which autoregulates its own synthesis. DnaA binds to sequences at oriC, and with energy from ATP causes open complex (ssDNA) to form. Other proteins are required, including HU, IHF, and RNA polymerase. The open complex is stabilized by SSB, and DnaB (helicase) binds with assistance of DnaC and ATP. Priming occurs (may utilize DnaG (primase) or RNA polymerase in vitro). DnaA protects initiator RNA from degradation by RNase H.

DNA methylation is also involved in origin of replication and DnaA production. Many GATC sequences at origin, and these are sites of dam methylation (N6 position of the A residue is methylated). Methylation affects binding of DNA to membrane, expression of dnaA gene, and initiation of replication. After replication oriC is hemimethylated; this form will bind to membrane, inhibiting dnaA expression as well as replication initiation, etc. After some time (mins), oriC detaches (why?) and is methylated. DnaA is synthesized and initiation occurs.

Replication proceeds bidirectionally from oriC to terC at 30-35 min on E. coli chromosome. Termination is bidirectional as well. One terminator blocks the advance of each replication fork; these are specialized sequences and require proteins such as Terminus utilization substance (Tus protein). At termination, the two chromosomes are concatenated (linked, like links of chain). DNA gyrase (and the closely related Topoisomerase IV (ParC/ParE proteins) are capable of deconcatenating the DNA loops. Partitioning of DNA also requires the MukB protein; function still not clear.

During TRANSCRIPTION, DNA information is copied into RNA information by dsDNA-dependent, RNA polymerase. Requires template dsDNA, rNTPs, and RNA polymerase

Reaction is biochemically simple, yet topologically complex.

RNA-P must: 1. locate proper start sites, 2. separate the DNA strands locally to read the base information on template strand, 3. move helically down template while releasing mRNA strand for translation by ribosomes, and 4. terminate transcription only at appropriate sites.

Catalytic core polymerase: four polypeptides [a2 b b']

RpoA = a, RpoB = b, RpoC = b'

In some species of bacteria, the RpoC polypeptide is divided into two parts: RpoC1 = g and RpoC2 = b'

The core polymerase is catalytically active and can synthesize RNA, but does not properly initiate synthesis at specific sites.

RNA polymerase Holoenzyme = Core + sigma factor = [a2 b b'] s = Es

E. coli has 6 known sigma factors (see Table); other bacteria (e.g., Bacillus subtilis) have even more than this. Eubacteria have two families of sigma factors (sigma-70 family related to RpoD of E. coli and sigma-54 family related to RpoN of E. coli).

RNA polymerase moves along template strand in the 3' to 5' direction while synthesizing RNA in the 5' to 3' direction.

RNA synthesis involves 3 steps: Initiation, Elongation, Termination

Initiation: binding of polymerase to promoter wit the formation of a stable [RNA-P]-[DNA] complex and catalysis of first 3' to 5' phosphodiester bond.

Elongation: translocation of RNA polymerase along the DNA template strand with synthesis of RNA

Termination: cessation of synthesis and dissociation of polymerase from DNA

The initiation point for RNA synthesis includes to DNA sequence motifs, the PROMOTER, that are highly conserved in all eubacteria (hence the term "consensus sequences or consensus promoter").

5' TTGACA--17 bp--TATAAT--5-6bp-(T/C)AT, where Ais the first nucleotide transcribed
       -35                           -10

The -10 sequence, so-called because it is centered about 10 nucleotides upstream from the start site for transcription, is also called the Pribnow box; the -35 sequence is called the recognition site. Recognition of the promoter = -35 + -10 sequence motifs, is achieved by the type-1 sigma factor (required for cell viability = sigma 70, s70, of E. coli).

Different types of sigma factors recognize different promoter sequences, and hence can change the pattern of gene expression of cell by altering the pattern of RNA synthesis.

During initiation, the RNA polymerase DNA complex must be converted from the "closed" complex (no unwinding of DNA strands) to the "open" complex (unwinding of about one turn of DNA helix from middle of the Pribnow box to just beyond the initiation site. The first nucleotide is usually a purine, most often an A residue (see above). Once first internucleotide bond is formed (5' pppPurine--pN-OH 3'), the initiation phase is completed.

"Promoter strength" refers to the frequency of initiation of transcription that occurs at a particular promoter sequence; "strong promoter" means frequent initiation--as much as once per second; "weak promoter" means one where transcription is initiated very infrequently--can be less than once per generation (dyanmic range is about 104).

After 8-9 nt are polymerized, sigma factor is released from RNA polymerase-DNA template complex; suggests conformational change. Sigma is free to bind to core and initiate new round of transcription initiation.

Elongation rate: 30-60 nt per sec (not uniform--depends on G+C content, sequence)

Elongation includes: NTP binding

bond formation between NTP and 3'-OH of growing RNA chain

pyrophosphate release

translocation of polymerase along DNA template

melting of DNA template

Several proteins affect elongation reactions.

NusA: multifunctional protein; doesn't bind to RNA-P when sigma is bound, and may bind to same site; it alters affinity for NTPs and accentuates pausing at certain sites, suggesting a regulatory role; also required for rRNA antitermination

NusB: enhances pausing, but also required for rRNA antitermination

NusG: enhance elongation, reduces pausing; required for rRNA antitermination; required for efficient function of Rho

GreA: can promote cleavage of 2-3 nt from 3' end of paused RNA-P complexes; release of pausing; editing function (?), since mismatching can cause pausing of RNA-P

GreB: can promote cleavage of up to 9 nt from 3' end; release of pausing

TERMINATION involves: 1. cessation of elongation; 2. release of transcript; 3. dissociation of RNA-P from termplate.

Several types of termination: 1. regulatory termination = ATTENUATION (not considered now)

2. SIMPLE = Rho-indendent (INTRINSIC) termination

3. COMPLEX = Rho-dependent termination

Characterized by G+C rich sequence with interrupted dyad symmetry followed by 4-8 A residues in template (or U residues in RNA). The dA residues cause arrest of the RNA polymerase; the stem-loop structures are critical for release of the RNA-P from template. [deoxy-A]-[ribo-U] hybrids are much less stable than other hybrids and have different structures in solution--could help trigger pausing and release

No particular consensus sequence, and binding sites are large (span about 150 to 200 bp of DNA). Rho is an RNA-dependent ATPase and has an RNA-DNA helicase activity (unwinds hybrid complex). Rho promotes termination at strong pause sites, but Rho also has sequence-specific binding to so-called "rut" sites in the RNA transcript. Rho prefers unpaired (ss) RNA rich in C residues. Rho binding probably confined to regions of transcripts not being translated; rRNA has secondary structure that blocks interaction (also bind r-proteins very readily).