CHAPTER 7 Essentials of Molecular Biology Genes and

CHAPTER 7 Essentials of Molecular Biology Genes and Gene Expression Informational macromolecules = DNA, RNA, Protein Unit of information = gene (a segment of DNA specifying a protein, r. RNA or t. RNA) Genes = coded by DNA or RNA (HIV) • The three key processes of macromolecular synthesis are: 1. DNA replication; • 2. transcription (the synthesis of RNA from a DNA template); and • 3. translation (the synthesis of proteins using messenger RNA as a template).

• Although the basic processes are the same in prokaryotes and eukaryotes, the organization of genetic information is more complex in eukaryotes.

• Most eukaryotic genes have both coding regions (exons) and noncoding regions (introns). Both introns and exons are transcribed into the primary transcript, an unprocessed RNA molecule that is the direct product of transcription.

DNA Structure: The Double Helix • DNA is a double-stranded molecule that forms a helical configuration and is measured in terms of numbers of base pairs. • Double stranded molecule is arranged in an antiparallel fashion. Purine = AG Pyrimidines = CT

• The two strands in the double helix are antiparallel. DNA size is expressed as bp, kbp or Mbp Size of E. coli DNA = 4640 kbp or 4. 64 Mbp

Inverted Repeats, Secondary Structure, and Stem-Loops Inverted repeats allow for the formation of secondary structure. Secondary structure, stem-loop is more common in RNA than in DNA

• The strands of a double-helical DNA molecule can be denatured by heat and allowed to reassociate following cooling (annealing and hybridization). s. DNA absorbed more uv than d. DNA

• In all cells, DNA exists as two polynucleotide strands whose base sequences are complementary. • The complementarity of DNA arises from the specific pairing of the purine (AG) and pyrimidine (CT) bases. • Adenine always pairs with thymine, and guanine always pairs with cytosine.

DNA Structure: Supercoiling • The very long DNA molecule can be packaged into the cell because it is supercoiled (Figure 7. 8). A break in a phophodiester bond (nick) changes a supercoiled molecule to a relaxed molecule

• In prokaryotes, this supercoiling is produced by enzymes called topoisomerases. In eukaryotic chromosomes, DNA is wound around proteins called histones, forming structures called nucleosomes.

• Topoisomerases - DNA gyrase is a key enzyme in prokaryotes, introducing negative supercoils to the DNA (Figure 7. 10). Reverse gyrase introduces positive supercoiling.

Chromosomes and Other Genetic Elements • In addition to the chromosome, a number of other genetic elements exist in cells.

• Plasmids are DNA molecules that exist separately from the chromosome of the cell. Mitochondria and chloroplasts contain their own DNA chromosomes. • Viruses contain a genome, either DNA or RNA, that controls their own replication. Transposable elements exist as a part of other genetic elements.

• Table 7. 2 shows the number, size, and configuration of chromosomes in a few microorganisms, both prokaryotic and eukaryotic.

DNA Replication • Both strands of the DNA helix serve as templates for the synthesis of two new strands (semiconservative replication).

• The two progeny double helices each contain one parental strand one new strand. The new strands are elongated by addition to the 3' end. 5’ (PO 4) 3’ (OH)

• DNA polymerases require a primer, which is composed of RNA (Figure 7. 13). E. coli polymerases = pol I-V Pol III is the primary enzyme for DNA synthesis It has 3 activities – 5’-3’ synthesis; 5’-3’ exo and 3’-5’ exonuclease Pol I has 5’-3’ synthesis and 5’-3’ exo (to remove RNA primers)

DNA Replication: The Replication Fork • In prokaryotes, DNA synthesis begins at a unique location called the origin of replication. • Table 7. 3 shows the major enzymes involved in DNA replication in Bacteria. The double helix is unwound by helicase and is stabilized by single-strand binding protein.

• As replication proceeds, the site of replication, called the replication fork, appears to move down the DNA. Okazaki fragment

• Extension of the DNA occurs continuously on the leading strand but discontinuously on the lagging strand (Figure 7. 15).

Sealing of nicks at the lagging strand

Errors in base pairing are corrected by proofreading functions associated with the activities of DNA polymerases. Pol I and III have 3’-5’ exonuclease that removes mismatched nucleotide Function of 5’-3’ activity?

Replication of prokaryotic chromosome • In Escherichia coli, and probably in all prokaryotes that contain a circular chromosome, replication is bidirectional from the origin of replication.

Replisome

Tools for Manipulating DNA Restriction Enzymes and Hybridization

• Restriction enzymes recognize specific short sequences in DNA and make breaks in the DNA. • Eco. R 1 cuts only unmodified DNA modified by Eco. R 1 methylase Palindrome

• The products of restriction enzyme digestion can be separated using gel electrophoresis Gel electrophoresis

Nomenclature

• The Southern blot (hybridization) technique is used to hybridize probes to DNA fragments that have been separated by gel electrophoresis to identify complementary sequences. Fragments complementary to the probe are circled yellow on the separation gel which hybridized to the probe.

Sequencing and Synthesizing DNA • DNA can be sequenced by the Sanger method, which involves copying the DNA to be sequenced in the presence of chain-terminating dideoxynucleotides

• The final products are separated by electrophoresis and the sequence is read. The short DNA primers required in this method can be synthesized chemically. Sequencing Methods Sanger method –(enzymatic, dideoxy chain termination) Dye-termination sequencing. This is a much more versatile method of sequencing, because it is not necessary to have a chemically modified oligonucleotide. The fluorescent dyes are conjugated to dideoxynucleotides, so a chain termination event is marked with a unique chemical group. Only one reaction needs to be run in this case, because there is no longer a separation between the label and the terminating group. Maxum and Gilbert method – (chemical degradation)

Synthesis of nucleotides for primers, probes and Site-directed mutagenesis Solid-phase procedure – First nucleotide is fastened to an insoluble porous support (50µ m silica gel)

Amplifying DNA: The Polymerase Chain Reaction • The polymerase chain reaction (PCR) is a procedure for amplifying DNA in vitro and employs a heat-stable DNA polymerase from thermophilic prokaryotes.

Heat (95 o. C) is used to denature the DNA into two single-stranded molecules, annealing of primers is achieved by reducing temp (70 o. C) and DNA synthesis (Primer extension) at (50 -60 o. C) in which each of the strand is copied by the polymerase. After each cycle, the newly formed double strands are again separated by heat, and a new round of copying proceeds. At each cycle, the amount of target DNA doubles. Annealing

• Applications of PCR • PCR is a extremely sensitive and specific and highly efficient method • Used in identifying organisms – 16 s. RNA analysis • Clinical diagnostics – to identify infectious agents • DNA fingerprinting – in forensic analysis to identify individuals • Gene expression studies – RT-PCR

RNA Synthesis: Transcription, • The three major types of RNA are messenger RNA (m. RNA), transfer RNA (t. RNA), and ribosomal RNA (r. RNA). • Transcription of RNA from DNA involves the enzyme RNA polymerase, which adds bases onto the 3' ends of growing chains. Unlike DNA polymerase, RNA polymerase needs no primer and recognizes a specific start site on the DNA called the promoter.

Transcription β, β’, α 2 - coreenzyme β, β’, α 2, σ – holoenzyme

EM

Diversity of Sigma Factors, Consensus Sequences, and Other RNA Polymerases

• In Bacteria, promoters are recognized by the sigma subunit of RNA polymerase. Promoters recognized by a specific sigma factor have very similar sequences. σ 70 is the major sigma factor in E. coli Heat-shock sigma factor Nitrate – dependent “ Flagella- specific gene “ Figure 7. 30 shows the sequence of a few promoters from Escherichia coli.

• In the Eukarya, the major classes of RNA are transcribed by different RNA polymerases, with RNA polymerase II producing most m. RNA pol I – most r. RNA pol II – all m. RNA pol III –t. RNA and one type of r. RNA • The single RNA polymerase of Archaea resembles RNA polymerase II in both structure and function. INR = Initiator element

Transcription Terminators • RNA polymerase stops transcription at specific sites called transcription terminators (Figure 7. 32). Rho dependent – rho causes termination by binding to m. RNA Intrinsic terminators – stem and loop structure with specific sequences poly U at 3’ and at stem.

• Although encoded by DNA, these signals function at the level of RNA. Some are intrinsic terminators and require no accessory proteins beyond the polymerase. In Bacteria, these sequences are often stem-loops followed by a run of U's. Other terminators require proteins, such as Rho.

The Unit of Transcription • Moncistronic vs. polycistronic m. RNA • The unit of transcription often contains more than a single gene. Transcription of several genes into a single m. RNA molecule may occur in prokaryotes, and so the m. RNA may contain the information for more than one polypeptide (Figure 7. 33).

• Genes that are transcribed together from a single promoter constitute an operon. In all organisms, genes encoding r. RNA are cotranscribed but then are processed to form the final r. RNA species.

Protein Synthesis - The Genetic Code • The genetic code is expressed in terms of RNA, and a single amino acid may be encoded by several different but related codons. • Table 7. 5 shows the genetic code as expressed by triplet base sequences of m. RNA. A codon is recognized following specific base-pairing with a sequence of three bases on a t. RNA called the anticodon.

• Some t. RNAs can recognize more than one codon. In these cases, t. RNA molecules form standard base pairs only at the first two positions of the codon, while tolerating irregular base pairing at the third position. This apparent mismatch phenomenon is called wobble (Figure 7. 34).

• A few codons, called nonsense codons, do not encode an amino acid. In addition to the nonsense codons, there is also a specific start codon that signals where the translation process should begin.

• It is important to have a precise starting point because with a triplet code, it is critical that translation begin at the correct location. If it does not, the whole reading frame will be shifted an entirely different protein (or no protein at all) will be formed (Figure 7. 35).

Transfer RNA • One or more transfer RNAs (Figure 7. 36) exist for each amino acid found in a protein. Enzymes called aminoacyl-t. RNA synthetases (Figure 7. 37) attach an amino acid to a t. RNA.

Aminoacyl t. RNA synthetases (one for t. RNAs of each a. a. )

• Once the correct amino acid is attached to its t. RNA, further specificity resides primarily in the codon-anticodon interaction.

Translation: The Process of Protein Synthesis • The ribosome plays a key role in the translation process, bringing together m. RNA and aminoacyl t. RNAs.

Shine-Dalgarno sequence (3 -9 nucleotides) Formylmethionine t. RNA – specific for start codon, AUG

• There are three sites on the ribosome: the acceptor site, where the charged t. RNA first combines; the peptide site, where the growing polypeptide chain is held; and an exit site. • During each step of amino acid addition, the ribosome advances three nucleotides (one codon) along the m. RNA, and the t. RNA moves from the acceptor to the peptide site. Termination of protein synthesis occurs when a nonsense codon, which does not encode an amino acid, is reached.

• During each step of amino acid addition, the ribosome advances three nucleotides (one codon) along the m. RNA, and the t. RNA moves from the acceptor to the peptide site. Termination of protein synthesis occurs when a nonsense codon, which does not encode an amino acid, is reached.

• Several ribosomes can translate a single m. RNA molecule simultaneously, forming a complex called a polysome.

Folding and Secreting Proteins • To function correctly, proteins must be properly folded. Folding may occur spontaneously but may also involve other proteins called molecular chaperones (Figure 7. 40).

Molecular chaprones Two systems in E. coli

• Many proteins also must be transported into or through cell membranes. Such proteins are synthesized with a signal sequence (Figure 7. 41) that is recognized by the cellular export apparatus and is removed either during or after export.