TAMIL Microbiology 222 Microbial Genetics (1+1)

TAMIL NADU AGRICULTURAL UNIVERSITY Dept of Agrl Microbiology

AGM 222 Microbial Genetics (1+1)

1 | P a g e II Year I Sem B. Tech (Biotech) and B. Tech (Bioinformatics) 2016‐17

Gene structure and Expression in bacteria and Eukaryotes Functionally the total genome of an organism may be divided into genes. Each gene is a sequence within the nucleic acid that represents a single protein. Each of the separate nucleic acid molecules comprising the genome may contain a large number of genes. Genomes for living organisms may contain as few as <500 genes (for a mycoplasma, a type of bacterium) to as many as >40,000 for Man.

The basic behavior of the gene was defined by Mendel more than a century ago. Summarized in his two laws, the gene was recognized as a "particulate factor" that passes unchanged from parent to progeny. A gene may exist in alternative forms. These forms are called alleles.

In diploid organisms, which have two sets of chromosomes, one copy of each chromosome is inherited from each parent. This is the same behavior that is displayed by genes. One of the two copies of each gene is the paternal allele (inherited from the father), the other is the maternal allele (inherited from the mother). The equivalence led to the discovery that chromosomes in fact carry the genes. A gene is a sequence of DNA that produces another nucleic acid, RNA. The DNA has two strands of nucleic acid, and the RNA has only one strand. The sequence of the RNA is determined by the sequence of the DNA (in fact, it is identical to one of the DNA strands). In many, but not in all cases, the RNA is in turn used to direct production of a protein. Thus a gene is a sequence of DNA that codes for an RNA; in protein‐coding genes, the RNA in turn codes for a protein. After the genetic information stored as DNA is transcribed into RNA, the information is translated to yield specific proteins. Collectively, these processes are called gene expression. The first stage is transcription, during which, an mRNA is produced using one strand of the DNA by the enzyme RNA polymerase (See a note on RNA Polymerase). The second stage is translation of the mRNA into protein. This is the process by which the sequence of an mRNA is read in triplets to give the series of amino acids that make the corresponding protein. RNA polymerase from Bacteria has five different subunits, designated as α, β, ω (omega) and σ (sigma) with β present in two copies. The β and β` (beta prime) subunits are similar but not identical. The subunits interact to form the active enzyme, called the RNA polymerase holoenzyme, but the sigma factor is not as tightly bound as the others and easily dissociates, leading to the formation of the RNA polymerase core enzyme - α2 β β` ω (minus sigma). The core enzyme alone synthesizes RNA, whereas the sigma factor recognizes the appropriate site on the DNA for RNA synthesis to begin. The omega subunit is needed for assembly of the core enzyme but not for RNA synthesis.


Transcription in Bacteria Transcription is carried out by RNA polymerase. Like DNA polymerase, RNA polymerase catalyzes the formation of phosphodiester bonds but between ribonucleotides rather than deoxyribonucleotides. RNA polymerase uses DNA as a template. The precursors of RNA are the ribonucleoside triphosphates ATP, GTP, UTP, and CTP. The mechanism of RNA synthesis is much like that of DNA synthesis. During elongation of an RNA chain, ribonucleoside triphosphates are added to the 3`‐OH of the ribose of the preceding nucleotide.

Transcription starts when RNA polymerase binds to a special region, the promoter, at the start of the gene. The promoter surrounds the first base pair that is transcribed into RNA, the start point. From this point, RNA polymerase moves along the template, synthesizing RNA, until it reaches a terminator sequence. This action defines a transcription unit that extends from the promoter to the terminator. The transcription unit constitutes a stretch of DNA expressed through the production of a single RNA molecule. A transcription unit may include more than one gene. Sequences prior to the startpoint are described as upstream of it; those after the startpoint (within the transcribed sequence) are downstream of it. Sequences are conventionally written so that transcription proceeds from left (upstream) to right (downstream). This corresponds to writing the mRNA in the usual 5'→ 3' direc on.


How RNA polymerse recognizes the start point of transcription RNA polymerase is a large protein and makes contact with many bases of DNA simultaneously. Proteins such as RNA polymerase can interact specifically with DNA because portions of the bases are exposed in the major groove. However, in order to initiate RNA synthesis correctly, RNA polymerase must first recognize the initiation sites on the DNA. These sites, called promoters, are recognized by the sigma factor.

How transcription stops? Only those genes that need to be expressed should be transcribed. Therefore it is important to terminate transcription at the correct position. Termination of RNA synthesis is governed by specific base sequences on the DNA, called Terminators.

In Bacteria a common termination signal on the DNA is a GC‐rich sequence containing an inverted repeat with a central nonrepeating segment. When such a DNA sequence is transcribed, the RNA forms a stem–loop structure by intra‐strand base pairing. Such stem–loop structures, followed by a run of adenosines in the DNA template and therefore a run of uridines in the mRNA, are effective transcription terminators. Polycistronic mRNA and the Operon

In prokaryotes, genes encoding related enzymes are often clustered together. RNA polymerase proceeds through such clusters and transcribes the whole group of genes into a single, long mRNA molecule. An mRNA encoding such a group of co‐transcribed genes is called a polycistronic mRNA. When this is translated, several polypeptides are synthesized, one after another, by the same ribosome.

A group of related genes that are transcribed together to give a single polycistronic mRNA is known as an operon. Assembling genes for the same biochemical pathway or genes needed under the same conditions into an operon allows their expression to be co‐ordinated. Despite this, eukaryotes do not have operons and polycistronic mRNA.


An Example for polycistronic mRNA. Here, the complete structure of ribosome is coded by 3 genes namely 16S, 23S and 5S. All these genes are under a common promoter and terminator. During transcription, RNA polymerase will synthesize a single mRNA (which is referred as polycistronic mRNA). After that the unwanted transcribed regions will be excised out and matured RNAs will be translated. However, these kind of post‐transcriptional processes are not common in bacterial system, while in eukaryotes, it is common.

In prokaryotes, most messenger RNAs have a short half‐life (on the order of a few minutes), after which they are degraded by cellular ribonucleases. This is in contrast to rRNA and tRNA, which are stable RNAs. Hence, the translation will be started immediately or even before the transcription is over. Transcription in Eukaryotes In Eukaryota, many genes are split into two or more coding regions separated by noncoding regions. The segments of coding sequence are called exons and introns are the intervening non‐coding regions. The term primary transcript refers to the RNA molecule that is originally transcribed before the introns are removed to generate the final mRNA, consisting solely of the exons. Another major distinguishable feature of eukaryotic gene expression is the physical separation of transcription (in nucleus) and translation (in cytoplasm).

During transcription, RNA is formed from a DNA template. The initial RNA product of transcription is known as the primary transcript. However, many RNA molecules need alterations ‐ known as RNA processing before they are mature, that is, ready to carry out their role in the cell. As we have seen, many genes in eukaryotes contain intervening sequences, the introns, between the protein‐coding regions, the exons. These intervening sequences are removed from the primary transcript. The RNA is cleaved to remove the introns and the exons are joined to form a contiguous protein‐coding sequence in the mature mRNA. The process by which introns are removed and exons are joined is called RNA splicing. Occasional introns are found in prokaryotes, but the mechanism of removal is different.


mRNA undergoes two modifications before splicing There are two other unique steps in the processing of eukaryotic mRNA. Both steps take place in the nucleus prior to splicing. The first, called capping, occurs before transcription is complete. Capping is the addition of a methylated guanine nucleotide at the 5`‐phosphate end of the mRNA. The cap nucleotide is added in reverse orientation relative to the rest of the mRNA molecule.

The second processing step consists of trimming the 3` end of the primary transcript and adding 100–200 adenylate residues as the poly(A) tail. The tail recognition sequence, AAUAAA, is located close to the 3` end of the primary transcript and beyond the stop codon of the protein encoded by the mRNA. (Thus the poly(A) tail is not translated). Spliceosome helps the splicing RNA splicing takes place in the nucleus. Splicing is done by a large macromolecular complex about the size of a ribosome, called the spliceosome. The spliceosome removes introns and joins adjacent exons to form mature mRNA. The spliceosome contains four large RNA–protein complexes, called small nuclear ribonucleoproteins (snRNPs), together with many protein factors; indeed, over 100 proteins participate in its activity. How spliceosome removes the introns is presented in this diagram. The poly(A) tail is also required for translation; it indicates to the translation machinery that the RNA is mRNA rather than some other form of RNA and that it is ready for translation. Only when all three steps (capping, poly(A) tailing and RNA splicing) complete is the mature mRNA transported into the cytoplasm for translation.

Translation in Prokaryotes and Eukaryotes Protein synthesis by eukaryotic ribosomes is generally more complex than in Bacteria. The cytoplasmic ribosomes of eukaryotic cells (80S ribosomes) are larger than bacterial ribosomes (70S ribosomes) and contain more rRNA and protein molecules. In particular, the large ribosomal subunit contains three rRNA molecules, 5S, 5.8S, and 28S. The 5.8S rRNA is homologous to the 5S end of bacterial 23S rRNA, and the 28S rRNA corresponds to the rest of bacterial 23S rRNA.

In Bacteria mRNA is polycistronic and may be translated to give several proteins. In eukaryotes, mRNA carries only a single gene that is translated into a single protein. That is, eukaryotic mRNA is monocistronic.


Bacteria use N‐formylmethionine as the first amino acid of all proteins, whereas eukaryotes use methionine.

The prokaryotic mRNA has ribosomal binding site which is absent in eukaryotic mRNA. Instead, the eukaryotic mRNA is recognized by its cap.

Over‐view of Translation

Comparative gene expression in bacteria and eukaryotes

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TAMIL Microbiology 222 Microbial Genetics (1+1)