Some basic molecular biology

Summaries of: Replication, Transcription; Translation, Hybridization, PCR

Material adapted from

Lodish et al, Molecular Cell Biology, 4th ed, Freeman, 2000; some figures from Genes VII by

Lewin, Oxford, 2000; Alberts et al, Essential Cell Biology, An Introduction to the Molecular Biology

of the Cell, Garland, 2000; and elsewhere.

DNA Replication

The regular pairing of bases in the double helical DNA structure suggested to Watson and Crick a mechanism of DNA synthesis, Their proposal that new strands of DNA are synthesized by copying of parental strands proved to be correct.

The DNA strand that is copied to form a new strand is called a template. The information in the template is preserved: although the first copy has a complementary sequence, not an identical one, a copy of the copy produces the original (template) sequence again. In the replication of double-stranded or duplex DNA molecule, both original (parental) DNA strands are copied. When copying is finished, the two new duplexes, each consisting of one of the original strands plus its copy, separate from each other.

DNA Replication, ctd All DNA synthesis proceeds in the same chemical direction: 5’->3’.

Nucleic acid chains are assembled from 5’ triphosphates of deoxyribonucleosides. Strand growth is energetically unfavourable, but is driven by the energy available in the triphosphates.

The enzymes that copy (replicate) DNA to make more DNA are DNA polymerases.

DNA polymerases cannot initiate chain synthesis de novo; instead they require a short preexisting DNA strand, called a primer, to begin chain growth. With a primer base-paired to the template strand, a DNA polymerase adds nucleotides to the free hydroxyl group at the 3’ end of the primer.

Replication of duplex DNA requires assembly of many proteins (at least 30) at a growing fork: helicases to unwind, primases to prime, ligases to ligate (join), topisomerases to remove supercoils, RNA polymerase, etc.

Transcription (mainly in prokaryotes) Transcription is a very complex process involving many steps and

many proteins.

• RNA polymerase initiates transcription of most genes at a unique position (a single base: the start site) in the template DNA upstream of the coding sequence.

• RNA polymerase binds to specific promoter sequences to initiate transcription

• Initiation begins when a subunit of a polymerase binds to a promotor DNA sequence

• Repressors bind to DNA sequences called operators which overlap the promoter region that contacts RNA polymerase. A bound repressor interferes with binding of RNA polymerase and transcription initiation

• Activators generally bind to DNA on the opposite side of the helix from the polymerase at specific positions

Transcription, ctd

• The activator cAMP-CAP stimulates transcription by forming a complex with RNA polymerase that has a greater affinity for DNA sites than the individual proteins

In eukaryotes, the situation is more complex still.

• Expression of eukaryotic protein coding genes is regulated through multiple transcriptional control regions

• Regulatory elements are often many kilobases from start sites

• There are three different types of RNA polymerases with different functions

• Enhancers, usually 100-200 bp in length contain multiple 8-20 bp control elements, may be from 200 bp to tens of kilobases upstream of downstream from a promoter, within an intron, or downstream from a final exon of gene

• Promoter-proximal elements and enhancers often are cell-type specific

.

From Alberts et al, 1998

From Genes VII

Translation• The AUG start codon is recognized by methionyl-tRNAi

Met

• Eukaryotic initiation of protein synthesis occurs at th 5’ end and internal sites in mRNA

• During chain elongation each incoming aminoacyl-tRNA moves through three ribosomal sites

• This process is catalyzed by proteins known as elongation factors

• Protein synthesis is terminated by a release factor when a stop codon is reached

• In eukaryotic cells, multiple ribosomes are commonly bound to a single mRNA, forming a circular polysome.

• As each ribosome completes translation and is released from the 3’ end of the mRNA, the subunits probably reassemble quickly at the 5’ end.

Hybridization

This is a technique which exploits a potent feature of the DNA duplex - the sequence complementarity of the two strands. Remarkably, DNA can reassemble with perfect fidelity from the separated strands. The melting temperature of a DNA molecule is denoted Tm and this depends on G+C content and salt concentration.

The Southern blotting technique

Polymerase Chain Reaction

This reaction is used to amplify specific DNA sequences in a complex mixture when the ends of the sequence are known. The source is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3’ ends of the segment of interest are added in great excess to the denatured DNA and the temperature is lowered to 50-60˚C or even lower. The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA.

PCR, ctd

The hybridized oligos then serve as primers for DNA chain synthesis, which begins upon addition of a supply of dNTPs and a temperature resistant polymerase such as that from Thermus aquilus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can extend primers at temperatures up to 72˚C. When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes. When the temperature is lowered again, a new round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify.