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.
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)
Essentials of Molecular Biology
Most eukaryotic genes have both coding regions ( and eukaryotes, the organization of genetic information is more complex in eukaryotes.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.
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.
DNA size is expressed as bp, kbp or Mbp and eukaryotes, the organization of genetic information is more complex in eukaryotes.
Size of E. coli DNA = 4640 kbp or 4.64 Mbp
The two strands in the double helix are antiparallel.
Inverted Repeats, Secondary Structure, and Stem-Loops and eukaryotes, the organization of genetic information is more complex in eukaryotes.
Inverted repeats allow for the formation of secondary structure.
Secondary structure, stem-loop is more common in RNA than in DNA
sDNA absorbed more uv than dDNA and eukaryotes, the organization of genetic information is more complex in eukaryotes.
The strands of a double-helical DNA molecule can be denatured by heat and allowed to reassociate following cooling (annealing and hybridization).
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.
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).
In prokaryotes, this whose base sequences are supercoiling is produced by enzymes called topoisomerases. In eukaryotic chromosomes, DNA is wound around proteins called histones, forming structures called nucleosomes.
Topoisomerases - whose base sequences are DNA gyrase is a key enzyme in prokaryotes, introducing negative supercoils to the DNA (Figure 7.10). Reverse gyrase introduces positive supercoiling.
In addition to the chromosome, a number of other genetic elements exist in cells.
Plasmids whose base sequences are 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 whose base sequences are shows the number, size, and configuration of chromosomes in a few microorganisms, both prokaryotic and eukaryotic.
Both strands of the DNA helix serve as templates for the synthesis of two new strands (semiconservative replication).
5’ (PO whose base sequences are 4)
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 whose base sequences are 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)
In prokaryotes, DNA synthesis begins at a unique location called the origin of replication.
Okazaki fragment whose base sequences are
As replication proceeds, the site of replication, called the replication fork, appears to move down the DNA.
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 strand but discontinuously on the lagging strand (
Errors strand but discontinuously on the lagging strand ( 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 strand but discontinuously on the lagging strand (
In Escherichia coli, and probably in all prokaryotes that contain a circular chromosome, replication is bidirectional from the origin of replication.
Palindrome strand but discontinuously on the lagging strand (
Restriction enzymes recognize specific short sequences in DNA and make breaks in the DNA.
EcoR1 cuts only unmodified DNAmodified by EcoR1 methylase
Gel electrophoresis strand but discontinuously on the lagging strand (
The products of restriction enzyme digestion can be separated using gel electrophoresis
Nomenclature strand but discontinuously on the lagging strand (
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.
DNA can be sequenced by the Sanger method, which involves copying the DNA to be sequenced in the presence of chain-terminating dideoxynucleotides
Sequencing Methods Sanger method the separation gel which hybridized to the probe. –(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 –
The final products are separated by electrophoresis and the sequence is read. The short DNA primers required in this method can be synthesized chemically.
Synthesis of nucleotides the separation gel which hybridized to the probe.
for primers, probes and
Solid-phase procedure – First nucleotide is fastened to an insoluble porous support (50µ m silica gel)
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 the separation gel which hybridized to the probe.oC) 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.
Applications of PCR the separation gel which hybridized to the probe.
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
The three major types of RNA are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).
Transcription the separation gel which hybridized to the probe.
β, β’, α2 - coreenzyme
β, β’, α2, σ – holoenzyme
EM the separation gel which hybridized to the probe.
In RNA PolymerasesBacteria, 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.
In the RNA PolymerasesEukarya, 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
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).
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.
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).
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.
The genetic code is expressed in terms of RNA, and a single amino acid may be encoded by several different but related codons.
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).
A few codons, called 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 (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 and an entirely different protein (or no protein at all) will be formed (Figure 7.35).
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.
Aminoacyl tRNA synthetases (one for tRNAs of each a. a.) 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 (
Once the correct amino acid is attached to its tRNA, further specificity resides primarily in the codon-anticodon interaction.
The ribosome plays a key role in the translation process, bringing together mRNA and aminoacyl tRNAs.
Shine-Dalgarno sequence (3-9 nucleotides) further specificity resides primarily in the codon-anticodon interaction.
Formylmethionine tRNA – specific for start codon, AUG
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.
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.
Several ribosomes 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. can translate a single mRNA molecule simultaneously, forming a complex called a polysome.
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 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.
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.