Fig. 5.9. Prokaryotes and Eukaryotes

Animation RNA processing

exon = expressed DNA sequences in a gene, code for amino acids. Introns and exons: Eukaryote pre-mRNAs often have intervening introns that must be removed during RNA processing (as do some viruses). intron = intragenic region made of non-coding DNA sequences between exons in a gene. exon = expressed DNA sequences in a gene, code for amino acids. Fig. 5.12 1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)

mRNA splicing of exons and removal of introns: Introns typically begin with a 5’-GU and end with AG-3’. Cleavage occurs first at the 5’ end of intron 1 (between 2 exons). The now free G joins with an A at a specific branch point sequence in the middle of the intron, using a 2’ to 5’ phosphodiester bond. Intron forms a lariat-shaped structure. Lariat is excised, and the exons are joined to form a spliced mRNA. Splicing is mediated by splicosomes, complexes of small nuclear RNAs (snRNAs) and proteins, that cleave the intron at the 3’ end and join the exons. Introns are degraded by the cell.

Fig. 5.12

Fig. 5.13

Animation Intron Removal

Significance of Introns: Introns are not junk DNA They have important regulatory functions. One of their primary attributes is that they enable the differential splicing of different exons, result in different protein variants with different functional properties. Multiple proteins from a single gene. These patterns of gene regulation are as important as the gene sequences themselves. They can also regulate the rate of transcription and function as a point for recombination. They can also be lost and gained, making them a type of mobile genetic element.

Post-transcriptional modification, mRNA editing: Adds or deletes nucleotides from a pre-RNA, or chemically alters the bases, so the mRNA bases do not match the DNA sequence. Can results in the substitution, addition, or deletion of amino acids (relative to the DNA template). Generally cell or tissue specific. Examples occur in protozoa, slime molds, plant organelles, and mammals.

Genes that do not code proteins also are transcribed: rRNA, ribosomal RNA Catalyze protein synthesis by facilitating the binding of tRNA (and their amino acids) to mRNA. tRNA, transfer RNA Transport amino acids to mRNA for translation. snRNA, small nuclear RNA Combine with proteins to form complexes used in RNA processing (splicosomes used for intron removal).

1. Synthesis of ribosomal RNA and ribosomes: Cells contain thousands of ribosomes. Consist of two subunits (large and small) in prokaryotes and eukaryotes, in combination with ribosomal proteins. E. coli 70S model: 50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins 30S subunit = 16S (1,542 nt) + 20 proteins Mammalian 80S model: 60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins 40S subunit = 18S (1,900 nt) + 35 proteins DNA regions that code for rRNA are called ribosomal DNA (rDNA). Eukaryotes have many copies of rRNA genes tandemly repeated.

Fig. 6.13, Mammalian example of 80S rRNA

Fig. 6.12

1. Synthesis of ribosomal RNA and ribosomes (continued): Transcription occurs by the same mechanism as protein-coding genes, but generally using RNA polymerase I. rRNA synthesis requires its own array of specific transcription factors (TFs) Coding sequences for RNA subunits within rDNA genes contain the following: internal transcribed spacer ITS external transcribed spacer ETS nontranscribed spacer NTS ITS units (analogous to introns) separate the RNA subunits through the pre-rRNA stage, whereupon ITS & ETS are cleaved out and rRNAs are assembled. Subunits of mature ribosomes are bonded together by H-bonds. Finally, transported to the cytoplasm to initiate protein synthesis.

Fig. 5.18 2nd edition

2. Synthesis of tRNA: tRNA genes also occur in repeated copies throughout the genome, and may contain introns. Each tRNA (75-90 nt in length) has a different sequence that binds a different amino acid. Many tRNAs undergo extensive post-transcription modification, especially those in the mitochondria and chloroplast. tRNAs form clover-leaf structures, with complementary base-pairing between regions to form four stems and loops. Loop #2 contains the anti-codon, which recognizes mRNA codons during translation (more about that when we discuss translation). Same general mechanism using RNA polymerase III, promoters, unique TFs, plus post-transcriptional modification from pre-tRNA.

Figure 6.9

3. Synthesis of snRNA (small nuclear RNA): Form complexes with proteins used in eukaryotic RNA processing, such as splicing of mRNA after introns are removed. Transcribed using RNA polymerase II or III. Associated with small nuclear ribonucleoproteins (snRNPs). Also function in regulation of transcription factors and maintenance of telomeres. U7 H/ACA