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CHAPTER 7 Essentials of Molecular Biology

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Genes and Gene Expression Informational macromolecules = DNA, RNA, Protein Unit of information = gene (a segment of DNA specifying a protein, rRNA or tRNA) Genes = coded by DNA or RNA (HIV). CHAPTER 7 Essentials of Molecular Biology.

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Genes and Gene ExpressionInformational macromolecules = DNA, RNA, ProteinUnit of information = gene (a segment of DNA specifying a protein, rRNA or tRNA)Genes = coded by DNA or RNA (HIV)

CHAPTER 7

Essentials of Molecular Biology

  • 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).
slide3

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

slide4

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 Structure: The Double Helix

Purine = AG

Pyrimidines = CT

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.

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

The two strands in the double helix are antiparallel.

slide8

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

slide9

sDNA absorbed more uv than dDNA

The strands of a double-helical DNA molecule can be denatured by heat and allowed to reassociate following cooling (annealing and hybridization).

slide10

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

DNA Structure: Supercoiling

A break in a phophodiester bond (nick) changes a supercoiled molecule to a relaxed molecule

The very long DNA molecule can be packaged into the cell because it is supercoiled (Figure 7.8).

slide14

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

slide15

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

Chromosomes and Other Genetic Elements

In addition to the chromosome, a number of other genetic elements exist in cells.

slide17

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.

slide18
Table 7.2 shows the number, size, and configuration of chromosomes in a few microorganisms, both prokaryotic and eukaryotic.
dna replication

DNA Replication

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

slide20

5’ (PO4)

3’ (OH)

The two progeny double helices each contain one parental strand and one new strand. The new strands are elongated by addition to the 3' end.

dna polymerases require a primer which is composed of rna figure 7 13
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

DNA Replication: The Replication Fork

  • 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.

In prokaryotes, DNA synthesis begins at a unique location called the origin of replication.

slide24

Okazaki fragment

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

slide25
Extension of the DNA occurs continuously on the leading strand but discontinuously on the lagging strand (Figure 7.15).
slide27

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?

slide28

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.

slide32

Palindrome

Restriction enzymes recognize specific short sequences in DNA and make breaks in the DNA.

EcoR1 cuts only unmodified DNAmodified by EcoR1 methylase

the products of restriction enzyme digestion can be separated using gel electrophoresis

Gel electrophoresis

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

slide35

Fragments complementary to the probe are circled yellow on the separation gel which hybridized to the probe.

The Southern blot (hybridization) technique is used to hybridize probes to DNA fragments that have been separated by gel electrophoresis to identify complementary sequences.

sequencing and synthesizing dna

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

slide37

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)

The final products are separated by electrophoresis and the sequence is read. The short DNA primers required in this method can be synthesized chemically.

slide38

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

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.

slide40

Heat (95oC) is used to denature the DNA into two single-stranded molecules, annealing of primers is achieved by reducing temp (70oC) and DNA synthesis (Primer extension) at (50-60oC) 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

slide41

Applications of PCR

PCR is a extremely sensitive and specific and highly efficient method

Used in identifying organisms – 16 sRNA analysis

Clinical diagnostics – to identify infectious agents

DNA fingerprinting – in forensic analysis to identify individuals

Gene expression studies – RT-PCR

rna synthesis transcription

RNA Synthesis: Transcription,

  • 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.

The three major types of RNA are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).

slide43

Transcription

β, β’, α2 - coreenzyme

β, β’, α2, σ – holoenzyme

slide46

In Bacteria, promoters are recognized by the sigma subunit of RNA polymerase. Promoters recognized by a specific sigma factor have very similar sequences.

σ70 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.

slide48

In the Eukarya, the major classes of RNA are transcribed by different RNA polymerases, with RNA polymerase II producing most mRNA.

RNA pol I – most rRNA

RNA pol II – all mRNA

RNA pol III –tRNA and one type of rRNA

The single RNA polymerase of Archaea resembles RNA polymerase II in both structure and function.

INR = Initiator element

transcription terminators

Transcription Terminators

Rho dependent – rho causes termination by binding to mRNA

Intrinsic terminators – stem and loop structure with specific sequences poly U at 3’ and at stem.

RNA polymerase stops transcription at specific sites called transcription terminators (Figure 7.32).

slide50

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

The Unit of Transcription

Moncistronic vs. polycistronic mRNA

The unit of transcription often contains more than a single gene. Transcription of several genes into a single mRNA molecule may occur in prokaryotes, and so the mRNA may contain the information for more than one polypeptide (Figure 7.33).

slide53

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

protein synthesis the genetic code

Protein Synthesis - The Genetic Code

  • Table 7.5 shows the genetic code as expressed by triplet base sequences of mRNA. A codon is recognized following specific base-pairing with a sequence of three bases on a tRNA called the anticodon.

The genetic code is expressed in terms of RNA, and a single amino acid may be encoded by several different but related codons.

slide56

Some tRNAs can recognize more than one codon. In these cases, tRNA 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).

slide58

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.

slide59

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 and an entirely different protein (or no protein at all) will be formed (Figure 7.35).

transfer rna

Transfer RNA

One or more transfer RNAs (Figure 7.36) exist for each amino acid found in a protein. Enzymes called aminoacyl-tRNA synthetases (Figure 7.37) attach an amino acid to a tRNA.

slide64
Once the correct amino acid is attached to its tRNA, further specificity resides primarily in the codon-anticodon interaction.
translation the process of protein synthesis

Translation: The Process of Protein Synthesis

The ribosome plays a key role in the translation process, bringing together mRNA and aminoacyl tRNAs.

slide66

Shine-Dalgarno sequence (3-9 nucleotides)

Formylmethionine tRNA – specific for start codon, AUG

slide68

During each step of amino acid addition, the ribosome advances three nucleotides (one codon) along the mRNA, and the tRNA 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.

There are three sites on the ribosome: the acceptor site, where the charged tRNA first combines; the peptide site, where the growing polypeptide chain is held; and an exit site.

slide69

During each step of amino acid addition, the ribosome advances three nucleotides (one codon) along the mRNA, and the tRNA 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.

slide70
Several ribosomes can translate a single mRNA molecule simultaneously, forming a complex called a polysome.
folding and secreting proteins

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).

slide72

Molecular chaprones

Two systems in E. coli

slide73

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.