Overview of gene expression: An RNA polymerase recognizes a promoter sequence in the DNA and transcribes an RNA. This molecule is further processed in eukaryotes to remove extra material inside the coding region (introns) but this is not necessary in prokaryotes. The resulting messenger RNA is translated into a polypeptide chain with the involvement of ribosomes, tRNAs and amino acids.
Amino acids are the component units of polypeptide chains. Amino acids contain an amino group, a carboxyl group, and a carbon in the middle with an "R" group of varying structure attached. The folding of a protein is determined by the nature of the R groups. Charged amino acids tend to be on the outside of the protein, neutral tend to be inside. Amino acids are joined during translation through peptide bonds.
A classic experiment demonstrated that the gene sequence and the amino acid sequence of the E. coli trpA gene were colinear. This involved both genetics (mapping mutants in the gene) and biochemistry (sequencing the protein).
RNA is the intermediate between the DNA and the protein. It differs from DNA in having a ribose sugar instead of deoxyribose, and having the base uracil instead of thymine. RNA polymerase is involved in transcribing an RNA copy off the DNA. It recognizes a promoter sequence, does not require a primer to start transcription and has chain growth at the 3’ end. In eukaryotes there are three types of RNA polymerase: pol I transcribes the large RNAs found in ribosomes, pol II transcribes most genes, and pol III transcribes various small RNAs including tRNAs, 5S RNA in ribosomes, and small RNAs involved in splicing. Termination of transcription involves specific sequences in the DNA and may also involve termination proteins.
Promoters have been characterized in three ways. (1) Comparing sequences of genes and regions "upstream" from the genes has allowed the TATA box (-10 upstream from start of transcription) and another box at —35 to be identified on the basis of conserved regions of sequence. The sequence most common to these regions is known as the "consensus sequence". (2) Studying mutants in the promoters has also provided insights into the importance of particular sequences. (3) Artificially producing changes in the promoter sequences ("reverse genetics") has also provided insights into the importance or particular sequences for promoter function.
RNA which is transcribed has sequences in addition to those coding for the amino acids. In prokaryotes, this involves extra material at the 5’ end (leader) and 3’ end (untranslated region, UTR). In eukaryotes, in addition to the leader and 3’ untransalted region, introns are present which are spliced out of the primary transcript. The existence of these was demonstrated through hybridization of the processed transcript to the viral DNA from which it was transcribed; loops were seen representing the introns. Other modifications to eukaryotic RNA include a 5’ G cap and a 3’ poly A tail. Eukaryotic mRNA on the whole is much longer-lived than prokaryotic mRNA. This may in part be due to stability imparted by the 3’poly A tail. The mechanics of removal of the introns involves consensus sequences which are recognized and a particle caled a spliceosome which contains protein plus RNA and which is involved in the splicing.
Characteristics of introns (1) More variable in sequence than coding regions, implying that the sequences generally are non-critical for function. (2) Tend to be large and more common in higher eukaryotes than in lower eukaryotes such as fruit flies and yeast. The BRCA1 gene is an example, with over 90% of the transcript being spliced away as 21 introns. (3) Introns often divide "domains" (folding/ functional regions) of proteins. This may relate to the origin of genes, since some genes are considered to possibly have evolved by joining parts / domains from different genes together ("exon shuffling" model). (4) Introns may have an ancient origin. This is supported by the conservation of intron location for many genes between plants and animals, suggesting that they existed prior to the divergence of plants and animals. Some believe that the ancestors of bacteria may have had introns and that they were secondarily lost in present-day bacteria.
Translation in prokaryotes is often coupled with transcription, meaning that the two take place in close proximity to each other (see Fig. 9.32). In contrast, in eukaryotes, transcription takes place in the nucleus and translation takes place in the cytoplasm.