Protetn

Extein Z

Extein l

Intein 1

Tntein Z

Chromosome with split dnaE gene

Cells that delete the intein DNA are killed by the intein protein.

existence of the intein. If a mutation occurs that deletes the intein DNA sequence from the middle of the host gene, the previously formed intein DNase cuts the cell's DNA at this point—a potentially lethal move. Thus, any cell with a single copy of DNA that deletes the useless intein DNA will be killed by the intein protein. Only cells that keep the intein survive. Inteins appear unnecessary for cell survival and intein-encoding DNA may be therefore be regarded as a form of selfish DNA. The origin of inteins is obscure.

In eukaryotes there are two copies of each gene. If one copy loses the intein DNA sequence, it will be cut into two fragments by the intein DNase. Yeast and many other eukaryotes can mend double-stranded breaks in their DNA by a special form of recombination. The second (undamaged) copy of the gene where the break occurred lends one strand of its DNA to mend the break. After the single stranded regions are filled in, the result is a repaired copy of the gene that is identical to the undamaged copy. Now both copies again have the intein DNA sequence inserted. This type of repair process is known as gene conversion (Fig. 12.20).

Although most inteins have DNase activity, some shorter inteins exist that do not. Possibly they are defective and have lost the original sequences that encoded the nucle-ase. Inteins are not alone in their attempts to kill cells that delete their encoding DNA sequence. Certain introns use the same tactic. In this case, the DNA of the intron is not just meaningless, but encodes a DNase that cuts in two any copies of the host gene that have lost the intron. Certain plasmids also have mechanisms to kill any cell that loses the plasmid. They use a different and more complicated approach, as plasmids are not inserts in host DNA and so there is no insertion site to recognize. In all such cases, the idea is that only host cells that carry the selfish DNA will survive.

gene conversion Recombination and repair of DNA during meiosis that leads to replacement of one allele by another. This may result in a non-Mendelian ratio among the progeny of a genetic cross

FIGURE 12.20 Gene Conversion Repairs Broken Chromosomes in Eukaryotes

A double-stranded break in one member of a pair of chromosomes can be repaired. Repair makes use of the intact chromosome and involves base pairing of the fragmented DNA with the intact DNA. After recognition, the base pairs in the gap are filled in and, when complete, the chromosomes separate.

^Chromosome ___copy 1

D°uble ---Chromosome stranded _, copy 2

break

FIGURE 12.20 Gene Conversion Repairs Broken Chromosomes in Eukaryotes

A double-stranded break in one member of a pair of chromosomes can be repaired. Repair makes use of the intact chromosome and involves base pairing of the fragmented DNA with the intact DNA. After recognition, the base pairs in the gap are filled in and, when complete, the chromosomes separate.

Modified bases are freque ntly found in tRNA and rRNA.

Short RNA guide molecules are used to locate the sites for base modification in eukaryotic rRNA.

Base Modification of rRNA Requires Guide RNA

RNA molecules often contain modified bases. These are made by chemical modification of pre-existing bases. This is especially true of tRNA, which contains a relatively high percentage of many different modified bases (Ch. 8). However, ribosomal RNA has several modified bases and even occasional mRNA molecules may have a modified base or two.

In the case of tRNA, individual enzymes are sufficient for the various base modifications. These recognize particular bases in specific regions of various tRNA molecules and modify them. This scenario also applies to bacterial rRNA, which is modified in only a handful of locations. In the case of eukaryotic rRNA, modification occurs at multiple sites and requires small RNA molecules in addition to the modification enzymes. These small RNA molecules are needed to locate the correct sites for modification, which they do by base pairing over a short region with the rRNA. Synthesis and processing of rRNA in eukaryotes occurs in the nucleolus and so the guide RNAs are known as small nucleolar RNAs (snoRNAs).

nucleolus Region of the nucleus where ribosomal RNA is made and processed

Base Modification of rRNA Requires Short Guide RNA 323

FIGURE 12.21 Recognition of Modification Sites on rRNA by snoRNA

The site on the rRNA to be modified is identified by base pairing to specific sequence on the snoRNA. After snoRNA/rRNA pairing, the methylase binds to the BoxC and BoxD sequences and then methylates one of the bases of the rRNA.

Nucleotides in eukaryotic rRNA are modified by methylation of the 2'-OH group of the ribose or by converting uridine to pseudouridine. Although the number of different types of modification is limited, the number of sites is very large. Thus human pre-rRNA is methylated at 106 positions and pseudo-uridylated at 95. The base sequences around the modification sites are rarely related and so there is no consensus sequence for a modifying enzyme to use. Instead there is a different snoRNA for each modification site. Each snoRNA is 70-100 nucleotides long and has a unique sequence that recognizes the modification site on the rRNA. In addition, all snoRNAs that recognize potential methylation sites share a sequence motif that is recognized by the methylase that modifies the bases (Fig. 12.21). Similarly, the family of snoRNAs that recognize pseudo-uridylation sites have a motif that binds the pseudo-uridylation enzyme.

Interactions between rRNA and snoRNA often involve G-U base pairing. This non-standard base pair is stable in dsRNA and also occurs in the base pairing between the guide RNA and mRNA in RNA editing (see below).

Since there are many base modifications there are several hundred different snoRNAs per cell in eukaryotes. Only a few snoRNAs are transcribed from standard genes. Most snoRNAs are encoded in the introns of other genes. The snoRNA is made by cutting up the intron after it has been spliced out of the mRNA (Fig. 12.22). Many of the genes whose introns contain snoRNA sequences encode ribosomal components, e.g. U16 snoRNA is encoded by part of intron 3 from the gene for L1 ribosomal protein.

dsRNA Double-stranded RNA pseudouridine An isomer of uridine that is introduced into some RNA molecules by post-transcriptional modification

Base Modification of rRNA Requires Short Guide RNA 323

FIGURE 12.21 Recognition of Modification Sites on rRNA by snoRNA

The site on the rRNA to be modified is identified by base pairing to specific sequence on the snoRNA. After snoRNA/rRNA pairing, the methylase binds to the BoxC and BoxD sequences and then methylates one of the bases of the rRNA.

Methylated bases and pseudouridine are the most common modifications in eukaryotic rRNA.

Many guide RNA molecules (snoRNA) are encoded inside introns.

FIGURE 12.22 Generation of snoRNA from Intron

After splicing the mRNA, the intron assumes a lariat structure. The snoRNA is spliced from the intron, leaving smaller fragments of RNA.

_RNA Exon Intron Exon

Splicing mRNA Exon Exon

CLEAVAGE / \

snoRNA

RNA editing involves changing the actual base sequence of messenger RNA.

RNA Editing Involves Altering the Base Sequence

Perhaps the most bizarre modification that messenger RNA may undergo is the alteration of its base sequence—known as RNA editing. Altering bases within the coding sequence of mRNA usually changes the final protein product that will be obtained. Perhaps not surprisingly RNA editing is very rare in most organisms. In mammals RNA editing is restricted to base substitution and may consist of C-to-U or A-to-I (inosine) changes and relatively few specific cases of mRNA editing are known. In plants both C to U and U to C editing occur quite frequently. More radical editing of mRNA occurs in certain protozoa where bases are inserted and deleted.

An example of C-to-U editing occurs in the human gene for apolipoprotein B, which encodes a protein of 4,563 amino acids, one of the longest polypeptide chains on record.

The full-length protein, apolipoprotein B100, is made in liver cells and secreted into the bloodstream. ApoB100 is required for the assembly of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles which carry lipids, including cholesterol, around the body. A short version with only 2,153 amino acids, apolipoprotein B48, is made by intestinal cells. It is secreted into the intestine where it plays a role in the intestinal absorption of dietary fats. The shorter apoB48 lacks the region of apoB100 (between residues 3,129 to 3,532) that is bound by the LDL receptor. Consequently, dietary fat carried by apoB48 is largely absorbed by the liver whereas VLDL or LDL particles with apoB100 can deliver cholesterol to peripheral tissues whose cells possess the LDL receptor.

RNA editing Changing the coding sequence of an RNA molecule after transcription by altering, adding or removing bases

RNA Editing Involves Altering the Base Sequence 325

FIGURE 12.23 Editing of Apolipoprotein B mRNA

A) The apolipoprotein B gene normally makes apolipoprotein B100 in the liver. B) In the intestine the mRNA is altered by base editing to make apolipoprotein B48. A deaminase binds to a CAA codon in association with accessory proteins. C) The cytosine in the CAA codon is converted to uracil giving UAA. D) The UAA serves as a new stop codon that halts translation in the middle of the coding region. This results in a truncated protein, apolipoprotein B48.

The short version, apoB48, is encoded by the same gene as apoB100 and is made by editing the mRNA. The CAA codon (for glutamine) at position 2,154 is changed to a UAA stop codon by an enzyme that deaminates this specific cytosine, so converting it to uracil. Several accessory proteins are needed to make sure that the deaminase binds only at the correct site (Fig. 12.23).

Although only a single gene is needed to produce two versions of apolipoprotein B, editing needs several extra proteins to recognize the editing site and convert the C to U. Having two apolipoprotein B genes of different lengths would surely be more economical and there is no known reason why the more complicated procedure of mRNA editing is used.

A-to-I editing of mRNA also occurs in mammals. In this case adenosine is converted by deamination into inosine by dsRNA adenosine deaminase. Recognition is due to formation of double-stranded regions by base pairing between the modification sites and sequences from neighboring introns. Thus, the intron sequence affects the final coding sequence of the mature mRNA. Consequently, this editing must occur before removal of the introns. The inosine generated acts as guanosine during translation and therefore A-to-I editing may change the sequence of the resulting protein if it occurs within a coding region. This happens in the mRNAs for both the glutamate and serotonin receptor proteins of the mammalian nervous system. Defects in editing result in severe neurological symptoms. A-to-I editing also occurs in the non-coding regions of a significant number of other genes. The effects of these changes are for the most part still uncertain.

deaminase An enzyme that removes an amino group

Start codon

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