L

Synthesis of a second strand of dna

New complementary strand of DNA

FIGURE 16.07 Viruses may Use Rolling Circle Replication

Rolling circle replication occurs as described in the previous figure (A), but the replication continues around the circular DNA (purple) for many rounds (B). In some viruses, the long single-stranded piece of DNA is cut and packaged into virus particles as single-stranded DNA. In other viruses, a complementary strand is synthesized, giving double-stranded DNA (C). The double-stranded segments are then cut and packaged as single genome units.

during the cell cycle. The regulation of replication is much better understood for multicopy plasmids than for single-copy plasmids.

The most interesting aspect of copy number regulation is the involvement of antisense RNA to control the initiation of plasmid replication. The details are best investigated for the multicopy plasmid ColE1, but the principle of using antisense RNA applies to single-copy plasmids also.

Initiation of ColE1 replication starts with the transcription of an RNA molecule of 555 bases that can act as a primer for DNA synthesis. This pre-primer RNA (sometimes called RNAII) is cleaved by ribonuclease H to generate a primer with a free 3'-OH group, which can be used by DNA polymerase I (Fig. 16.08).

antisense RNA An RNA molecule that is complementary to messenger RNA or another functional RNA molecule ribonuclease H A ribonuclease of bacterial cells that is specific for RNA-DNA hybrids

FIGURE 16.08 Priming of ColEl Plasmid Replication

RNA poly merase synthesizes a strand of RNA (RNAII) near the origin of replication. RNAII (blue strand) is recognized and cleaved by ribonuclease H. The free 3'-OH created by the cleavage primes the synthesis of DNA at the origin. The ColE1 plasmid is then replicated.

FIGURE 16.08 Priming of ColEl Plasmid Replication

RNA poly merase synthesizes a strand of RNA (RNAII) near the origin of replication. RNAII (blue strand) is recognized and cleaved by ribonuclease H. The free 3'-OH created by the cleavage primes the synthesis of DNA at the origin. The ColE1 plasmid is then replicated.

If ribonuclease H fails to cleave the pre-primer RNAII, no free 3'-end is made and replication cannot proceed. Ribonuclease H is specific for RNA-DNA hybrids. Consequently, when an antisense RNA, known as RNAI, binds to pre-primer RNAII, this prevents cleavage (Fig. 16.09). Both RNAII and RNAI are transcribed from the same region of DNA but in opposite directions. RNAI is 108 bases long and is encoded

Plasmid Addiction and Host Killing Functions 435

pRE-PRIMER RNA FOLDS UP

FIGURE 16.09 Antisense RNA Prevents Primer Formation

A second transcript, RNAI, is also made from the same region of ColE1 as RNAII. The RNA I (green) is transcribed from the opposite DNA strand and is therefore complementary to RNAII. The complementary regions of RNAII and RNAI start to base pair, forming a region of bubbles. Eventually, the entire sequence aligns and a double stranded RNA molecule is formed. Since ribonuclease H only recognizes DNA-RNA hybrid molecules, no cut is made and RNAII transcription continues. DNA synthesis fails to start and the ColE1 plasmid is not replicated.

Base pairing starts in loop regions

Antisense RNA binds to pre-primer RNA

Conformation changes

RNA is not cleaved by RNase H

RNAs bind more tightly

Transcription continues

RNA is not cleaved by RNase H

RNAs bind more tightly by the opposite strand from RNAII. RNAI is complementary to the 5'-end of RNAII.

The copy number is determined by the relative strengths of binding of RNAII to the DNA at the origin of replication and of RNAI to RNAII. Mutations affecting either of these interactions will change the copy number. The Rom protein, also encoded by a gene on ColEl, increases binding between RNAI and RNAII. If the gene for Rom protein is inactivated, the copy number rises, but is still controlled.

Large plasmids often make toxins that kill the host cell if, and only if, it loses the plasmid DNA.

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