Transcription and Translation he conventional concept of central dogma of life which in essence is “DNA makes RNA makes protein’ is an oversimplitication of molecular biology. With the advances in cell biology and rapid developments in bic- informatics, the terms genome, transcriptome and proteome are in current use to represent the central dogma of molecular biology \Fig.25.1), Some information on the new concepts and terminology is given hereunder: GENOME The total DNA {genetic information) contained in an organism or a cell is regarded as the genome, Thus, the genome is the starchouse of biological information. it includes the chromosomes in the nucleus and the DNA in mitochondria, and chloroplasts Genomics : The study of the structure and function of genome is genomics. The term functional genomics is used to represent the ene expression and relationship of genes with ene products. Structural genomies refers to the Centeatn Replication [onal (GENOME? OTe [anal TD ‘Translation J ereiton 4 PROTEW PRE oA Conventional concept Current concept (pte-biointormatcs era) (bioinformatics era) Fig. 25.1 : The central dogma of ile (or molecular ‘ology) represented inthe form of conventional ‘and current concepts. structural motifs and complete protein structures. Comparative genomics ‘nvolves the study of comparative gene function and phylogeny TRANSCRIPTOME The RNA copies of the active protein coding genes represent transcripiome. Thus, transcriptome is the initial product of gene 542Chapter 25 «TRANSCRIPTION AND TRANSLATION 343 expression which directs the synthesis of, proteins. Transcriptomics : The study of transcriptome that involves all the RNA molecules made by a Cell, tissue oF an organism is transcriptomics PROTEOME The cell's repertoire (repository/storehouse) of proteins with their nature and biological functions is regarded as proteome. Thus, proteame represents the entire range of proteins and their biological functions in a ceil Proteomics : The study of the proteome. Metabolomics : The use of genome sequence analysis for determining the capability of a cell, tissue or an organism to synthesize small molecules {metabolites} is metabolomics. Whether the central dogma of life is represented in the conventional or more recent form, replication, transcription and translation are the key or core processes that ultimately control life. Replication of DNA has been described in Chapter 24, while transcription and translation are discussed in this chapter. Rese Transcription is a process in which ribo- nucleic acid (RNA) is synthesized from DNA. The word gene refers ‘© the functional unit of the DNA that can be transcribed. Thus, the xenetic information stored in DNA is expressed through RNA. For this purpose, one of the two strands of DNA serves as a template inon-coding, strand of sense strand) and produces working copies of RNA molecules. The other DNA strand which does not participate in transcription is, referred to as coding strand or antisense strand {frequently referred to as coding strand since with the exception of T for U, primary m&NA contains codons with the same base sequence). Transcription is selective The entire molecule of DNA is not expressed in transcription. RNAs are synthesized only for © Core enzyme Sigma factor aa Holoenzyme Fig. 25.2: RNA polymerase of & co some selected regions of DIA. For certain other regions of DNA, there may not be any transcription at all, The exact reason for the selective transcription is not known. This may be due to some inbuilt signals in the DNA molecule, The product formed in transcription is referred to as primary transcript. Most often, the primary RIA transcripts are inactive, They undergo certain alterations (splicing, terminal additions, base modifications etc.) commonly known as post-transcriptional modifications, to produce functionally active RNA molecules There exist certain differences in the transcription between prokaryotes and eukaryotes. The RNA synthesis in prokaryotes is given in some detail. This is followed by a brief discussion on eukaryotic transcription TRANSCRIPTION IN PROKARYOTES ‘A single enzyme—DNA dependent RNA polymerase or simply RNA polymerase— synthesizes all the RNAs in prokaryotes. RNA polymerase of F. coli is a complex holoenzyme {mol wt. 465 kDa) with five polypeptide subunits—2ay, 1B and 1’ and one sigmals) factor (Fig.25.2). The enzyme without sigma factor is referred to as core enzyme (cpp) ‘An overview of RNA synthesis is depicted in Fig.25.3. Vranscription involves three different stages—initiation, elongation and_ termination (Fig.25.4),544 BIOCHEMISTRY SANA FSeding strand E Template ; femplate RNA polymerase jation The binding of the enzyme RNA polymerase to DNA is the prerequisite for the transcription to start. The specific region on the DNA where the enzyme binds is known as promoter region. There are two base sequences on the coding DNA strand which the sigma factor of RNA polymerase can recognize for initiation of transcription (Fig.25.5). 1. Pribnow box (TATA box) : This consists of 6 nucleotide bases (TATAAT}, located on the left side about 10 bases away (upstream) from the starting point of transcription. 2, The ‘35! sequence : This is the second recognition site in the promoter region of DNA It contains a base sequence TTGACA, which is located about 35 bases (upstream, hence ~35) away on the left side from the site of transcription start Elongation ‘As the holoenzyme, RNA polymerase recognizes the promoter region, the sigma factor is released and transcription proceeds. RNA is synthesized from 5’ end to 3’ end i3/>3') antiparallel to. the DNA template. RNA polymerase utilizes ribonucleotide triphosphates (ATP, GTP, CTP and UTP) for the formation of RINA. For the addition of each nucleotide to the growing chain, a pyrophosphate moiety is released The sequence of nucleotide bases in the mRNA is complementary to the template DNA strand. It is however, identical to that of coding strand except that RNA contains U in place of T in DNA (Fig.25.6), RNA polymerase differs from DNA polymerase in two aspects. No primer is required for RNA polymerase and, further, this enzyme does not possess endo- or exonuclease activity Due to lack of the latter function (proof-reading, activity), RNA polymerase has no ability to repair the mistakes in the RNA synthesized. This is in contrast to DNA replication which is carried out with high fidelity, It is, however, fortunate that mistakes in RNA synthesis are less dangerous, since they are not transmitted to the daughter cells The double helical structure of DNA unwinds {as the transcription goes on, resulting in supercoils. The problem of supercoils is overcome by topoisomerases (more details in Chapter 24) Termina The process of transcription stops by termination signals. Two types of termination are identified. 1. Rho (p) dependent termination : A specific protein, named p factor, binds to the growing RNA (and not to RNA polymerase) or weakly to DNA, and in the bound state it acts as ATPase and terminates transcription and releases RNA The p factor is also responsible for the dissociation of RNA polymerase from DNA. 2. Rho (p) independent termination : The termination in this case is brought about by the formation of hairpins of newly synthesized RNA This occurs due to the presence of palindromes, A palindrome is a word that reads alike forward and backward e.g. madam, rotor. The presence of palindromes in the base sequence of DNA template (same when read in opposite direction) in the termination region is known. As a result of this, the newly synthesized RNA folds to form hairpins {due to complementary base pairing) that cause termination of transcription} * | ee of \ r i ° oy BLS] HE Keo re Ai fa Ve a Bf fe nel oq fot mae \ a iE | . Bo : : a =546 BIOCHEMISTRY 35 Prionow Sequence ‘box Coding 5 ATAAT: stand Template stan sears : Som, [sane | eg Seon Start of transcription ATGCAT TACGTACCET GCA © Coding strand 5) Template strand tionship. TRANSCRIPTION IN EUKARYOTES RIA synthesis in eukaryotes is # much more complicated than the transcription described above for prokaryotes. As such, ail the details of eukaryotic transcription (particularly about termination) are not clearly known. The salient features of available information are given hereunder RNA polymerases The nuclei of eukaryotic ceils possess three distinct RNA polymerases (Fig.25.7) 1. RNA polymerase I is responsible for the synthesis of precursors for the large ribosomal RIAs 2. RNA polymerase Il synthesizes the fecursors for mRNAs and small nuclear RNAS. 3. RNA polymerase It participates inthe formation of RNAs and smal! ribosomal RNAS. Besides the three RNA polymerases found in the nucleus, there also exists a mitochondrial RNA polymerase in eukaryotes. The latter resembles prokaryotic RNA polymerase in structure and function, Promoter sites In eukaryotes, a sequence of DNA bases— which is almost identical to pribnow box of prokaryotes—is identified (Fig.25.8). This sequence, known as Hogness box (or TATA box) t I NA polymerase | FRNA polymerase i | ae Ge ane ee Cal aoe) Fitesomal aa AN INAS: in eukaryotesDifference between prokaryotic and eukaryotic transcri| LOCATION PROCESS TRANSCRIPTION Cul) TRANSLATION ir POLYMERASE PROMOTERS RHO FACTOR Patol Ten Was POST TRANSCRIPTIONAL MODIFICATIONS Occurs in the cytoplasm. Prokaryotic transcription is not as complicated as eukaryotic transcription. Transcription and translation are coupled. In prokaryotic transcription, single kind of RNA polymerase is involved. Prokaryotic promoters iat) CORLL 1S (ea Termination of transcription needs a Rho factor in prokaryotes. Are polycistronic Do not occur Occurs in the nucleus. The transcription in a Pret eta Nein tent more complicated. Transcription and translation occur ere 4 Eukaryotic cell has three different kinds of RNA polymerases. Eukaryotic promoters have more variation than prokaryote promoters. _ Eukaryotes do not need Rho factors. Are monocistronic Occur during eukaryotic transcription TRANSCRIPTIONAL Needs an additional set FACTORS : ae irelcetaene lee) " transcriptional factors to bind RNA polymerase to the promoter, which are not a part of RNA polymerase. Needs sigma factor to bind to the promoter.