FCH 532 Lecture 4a

Chapter 5: Molecular biology overview

Transcription• Catalyzed by RNA polymerase.• Couples NTPs (ATP, CTP, GTP, UTP) to make RNA

• (RNA)n residues + NTP (RNA)n+1 residues + P2O74-

• 5’ 3’ nucleotides are added to the free 3’-OH group• Nucleotides must meet Watson-Crick base pairing

requirements with the template strand

Figure 5-23 Action of RNA polymerases.

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Transcription

• Transcribes only one template DNA strand at a time.• RNA polymerase will move along the duplex DNA it is

transcribing and creates a transcription bubble• This forms a short DNA-RNA hybrid with newly

synthesized RNA.• DNA template strand is read 3’ 5’

Figure 5-24 Function of the transcription bubble.

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Transcription

• DNA template contains control sites consisting of specific base sequences that specify where the RNA polymerse initiates transcription and the rate of transcription.

• activators and repressors control the sites in prokaryotes.

• Transcription factors bind to these sites in eukaryotes.

• messenger RNA (mRNA) - RNAs that encode proteins

• Rates at which cells synthesize a protein are determined by the rate at which mRNA synthesis is initiated.

• Promoter-in prokaryotes-a sequence that precedes the transcriptional initiation site.

Transcription• Prokaryotes can control transcriptional initiation in complex

manners.• Example E. coli lac operon.• Has 3 consecutive genes (Z, Y, and A) that are necessary to

metabolize lactose.• In the absence of lactose, the lac repressor protein binds a

control site in the lac operon called an operator.• This prevents the RNA polymerase from initiating transcription.• If lactose is present, some of the lactose is converted to

allolactose which binds to the lac repressor causing it to fall of the operator sequence.

• This allows RNA polymerase to initiate transcription of the genes.

Figure 5-25 Control of transcription of the lac operon.

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Eukaryotic RNA undergoes post-transcriptional modification

• In order for mRNAs in eukaryotes to become functional, they must undergo modifications.

• 7-methylguanosine-containing “cap” is added to the 5’ end. 250 nucleotide polyadenylic acid [poly(A)] tail is added to

the 3’ end.• Undergo gene splicing in which RNA segments called introns

are excised from the RNA and the remaining exons are rejoined to form the mature mRNA.

Figure 5-26 Post-transcriptional processing of eukaryotic mRNAs.

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mRNA

• In prokaryotes, transcription and translation both take place in the cytosol.

• Prokaryotic mRNAs have a short lifetime (avg. 1-3 min). They are degraded by nucleases.

• Rapid turnover in prokaryotes allows the prokaryote to respond quickly to the environment.

• In eukaryotic cells, RNAs are transcribed and post-translationally modified in the nucleus, then sent to cytosol.

• Eukaryotic mRNAs have lifetimes of several days.

Translation: Protein synthesis• Polypeptides are synthesized from mRNA by ribosomes.• Ribosomes are 2/3 rRNA (ribosomal RNA) and 1/3 protein.• Prokaryote ribosomes approx. 2500 kD, eukaryotes 4300 kD

• Transfer RNAs (tRNAs) deliver amino acids to the ribosome.• mRNA sequences can be broken down to codons-

consecutive 3-nucleotide segments that specify a particular amino acid.

• Once the mRNA binds to the ribosome, they specifically bind to the tRNA that is covalently linked to an amino acid.

Figure 5-27 Transfer RNA (tRNA) drawn in its “cloverleaf” form.

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tRNA has 76 nucleotides

Has an anticodon-complementary sequence to the mRNA sequence

Amino acid is linked to the 3’ end of the tRNA to form aminoacyl-tRNA.

tRNAs are “charged” with amino acids by specific enzymes (aminoacyl-tRNA synthetases or aaRSs)

Figure 5-28 Schematic diagram of translation.

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Figure 5-29 The ribosomal reaction forming a peptide bond.

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Genetic code• Correspondence between the sequence of bases in a codon and the

amino acid residue it specifies.• Nearly universal.• 4 possible bases (U[T], C, A, and G) can occupy three positions of

codon, therefore 43 = 64 possible codons.• 61 codons specify amino acids, and three UAA, UAG, and UGA are

stop codons (cause ribosome to end polypeptide synthesis and release the transcript).

• All but two amino acids (Met, Trp) are specified by more than one codon.

• Three (Leu, Ser, Arg) are specified by six codons.• Synonyms-multiple codons can code the same amino acid.• tRNA may recognize up to 3 synonymous codons because the 5’

base of a codon and 3’ base of the anticodon can interact in ways other than via Watson-Crick base pairs.

• Translation is initiated at the AUG codon (Met) but this tRNA differs from the tRNA for internal amino acid the Met codon.

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Figure 5-30 Nucleotide reading frames.

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DNA replication

• DNA is replicated similar to RNA with some differences:• 1. Deoxynucleotide triphosphates (dNTPs) are used

instead of NTPs • 2. Enzyme is the DNA polymerase• Other differences:• RNA polymerase can link together two nucleotides on

DNA template, but DNA polymerase can only extend (in the 5’ to 3’) direction an existing polynucleotide that is base paired to the template strand.

• DNA polymerase needs an oligonucleotide primer to initiate synthesis.

• Primers are RNA.

Figure 5-31 Action of DNA polymerases.

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DNA strands replicated in different ways

• DNA strands are simultaneously replicated.• Takes place at replication fork - junction where the two

parental DNA are pried apart and where the two daughter strands are synthesized.

• Leading strand is continuously copied from the 3’ to 5’ parental template in the 5’ to 3’ direction

• Lagging strand is discontinuously replicated in pieces from the 5’ to 3’ parental strands.

Figure 5-32aReplication of duplex DNA in E. coli.

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Figure 5-32bReplication of duplex DNA in E. coli.

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DNA strands replicated in different ways

• DNA strands are simultaneously replicated.• Takes place at replication fork - junction where the two

parental DNA are pried apart and where the two daughter strands are synthesized.

• Leading strand is continuously copied from the 3’ to 5’ parental template in the 5’ to 3’ direction

• Lagging strand is discontinuously replicated in pieces from the 5’ to 3’ parental strands.

• E. coli has 2 DNA polymerases necessary for survival. DNA polymerase III (Pol III) synthesizes the leading strand and most of the lagging strand.

• DNA polymerase I (Pol I) removes RNA primers and replaces them with DNA. This enzymes also has a 5’ to 3’ exonuclease activity.

Figure 5-33 The 5 3 exonuclease function of DNA polymerase I.

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Figure 5-34 Replacement of RNA primers by DNA in lagging strand synthesis.

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Lagging strand synthesis

• Synthesis of the leading strand of DNA is completed by the replacement of the RNA primer by DNA.

• Lagging strand is completed after nicks between multiple disconinuously synthesized segments are sealed by DNA ligase.

• Catalyzes the links of 3’-OH to 5’-phosphate groups.

Figure 5-35 Function of DNA ligase.

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Errors in DNA sequence can be corrected

• RNA polymerase has an error rate of 1 in 104 base pairs in E. coli.

• Pol I and Pol III have 3’ 5’ exonuclease activities. • This activity degrades the newly synthesized 3’ end of a

daughter strand one nucleotide at a time to edit out mistakes that are sometimes incorporated.

• Other enzymes are present that detect and correct errors in DNA damage that occurs from UV radiation and mutagens (chemical substances that damage DNA) and hydrolysis.

Figure 5-36 The 35 exonuclease function of DNA polymerase I and DNA polymerase III.

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Molecular cloning

• Clone-a collection of identical organisms that are derived from a single ancestor.

• Molecular cloning techniques - genetic engineering, recombinant DNA technology.

• Main idea is to insert a DNA segment of interest into an automously replicating cloning vector so that the DNA segment is replicated with the vector.

• Cloning into a chimeric vector in a suitable host organism results in large amounts of the inserted DNA segment.

Restriction endonucleases• Restriction enzymes (endonucleases) cleave DNA at

specific sequences within a polynucleotide.• Bacteria use restriction/modification systems as a small

scale immune system for protection from infection by foreign DNA.

• W. Arber and S. Linn back in 1969 showed that plating efficiencies of bacteriophage lambda grown in E. coli strains C, K-12, and B were very different.

E. coli strain on which parental phage had been grown

E. coli strain for plating phage

C K-12 B

C 1 <10-4 <10-4

K-12 1 1 <10-4

B 1 <10-4 1

* The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases which were methylated. * Additional studies with other strains indicate that different strains had specific methylated bases. * Typical sites of methylation include the N6 position of adenine, the N4 position of cytosine, or the C5 position of cytosine.

* In addition, only a fractional percentage of bases were methylated (i.e. not every adenine was methylated, for example) and these occurred at very specific sites in the DNA. * A characteristic feature of the sites of methylation, was that they involved palindromic DNA sequences.

* In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities. * The endonucleases cleaved at or near the methylation recognition site.

* These specific nucleases, however, would not cleave at these specific palindromic sequences if the DNA was methylated.

Thus, this combination of a specific methylase and endonuclease functioned as a type of immune system for individual bacterial strains, protecting them from infection by foreign DNA (e.g. viruses).

* In the bacterial strain EcoR1, the sequence GAATTC will be methylated at the internal adenine base (by the EcoR1 methylase). * The EcoR1 endonuclease within the same bacteria will not cleave the methylated DNA. * Foreign viral DNA, which is not methylated at the sequence "GAATTC" will therefore be recognized as "foreign" DNA and will be cleaved by the EcoR1 endonuclease. * Cleavage of the viral DNA renders it non-functional.

Figure 5-37 Restriction sites.

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Types of restriction endonucleases

• Type I and Type II restriction enzymes have both endonuclease and methylase activity on the same polypeptide.

• Type I enzymes cleave the DNA at a location of at least 1000 bp away from the recognition sequence.

• Type III enzymes cleave DNA from 24 to 26 bp away from the recognition site.

• Type II restriction enzymes are separate from their methylases. These enzymes cleave DNAs at specific sites within the recognition sequence (table 5-4).

Restriction endonucleases recognize pallindromic sequences• Palindrome-a word, verse, or sentence that reads the

same forwards or backwards.

• Restrction enzymes cleave 2 DNA strands at positions that are symmetrically staggered about the center of the palindromic sequence.

• Yields restction fragments with complementary single-stranded ends (1-4 nt in length called sticky ends).

• The sticky ends can associate by complementary base pairing with other fragments generated by the same restriction enzyme.

Figure 5-37 Restriction sites.

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Restriction maps• After digest with DNA restriction endonuclease the fragments

can be separated according to size by gel electrophoresis.• DNA can be separated according to size by agarose or

polyacrylamide.• Duplex DNA is detected by staining with intercalating, planar,

aromatic cations such as ethidium, acridine orange, or proflavin, between stacked base pairs. New stains like SYBR are available that are notThese exhibit flurorescence under UV light.

• As little as 50 ng of DNA may be detected in a gel by staining it with ethidum bromide.

• Can also be used to visualize single stranded DNA or RNA.• Can be used to generate a restriction map.

Figure 5-38 Agarose gel electrophoretogram of restriction digests.

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23130 bp

9416 bp6557 bp

4361 bp

2322 bp2027 bp

Digest of Agrobacterium radiobacter plasmid pAgK84 digested with:

A. BamHI, B. PstI, C. BglII, D. HaeIII, E. HincII, F. SacI, G. XbaI, H. HpaI. Lane I contains l phage DNA digested with HindIII as standards.

Figure 5-39 Construction of a restriction map.

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Figure 5-40 Restriction map for the 5243-bp circular DNA of SV40.

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