Chapter 3. DNA, RNA, and Protein Synthesis

I. DNA and RNA structure

II. DNA replication

III. Genetic code

IV. Gene expression

Transcription

Translation

Regulation

I. Structure of nucleic acids

Deoxyribonucleic acid (DNA) Structure

Nucleotides

See Fig. 3.1

Components

1. 5-Carbon sugar = 2'-deoxyribose

2. Base

Adenine, Guanine, Thymine or Cytosine

3. Phosphate

Linkage of Nucleotides

See Fig. 3.2

Phosphodiester bonds link nucleotides to form a single strand of DNA

A strand of DNA has 2 different ends

5' end contains a phosphate group

3' end contains a hydroxyl group

DNA Structure - A Double Stranded Helix

See Fig. 3.4

DNA in cells consists of two strands

The two strands are antiparallel (run in opposite directions)

5'------------->3'
3'<-------------5'

Strands are held together by hydrogen bonds between the bases

A hydrogen bonds only to T

G hydrogen bonds only to C

 

A and T are held together by 2 hydrogen bonds

G and C are held together by 3 hydrogen bonds

 

A molecule of DNA is helical (looks like a twisted ladder)

The order of the bases of a strand determines the sequence of the strand

The sequence is read starting from the 5' end

E.g.

5' T-A-T-G-C-G 3'

3' A-T-A-C-G-C 5'

The sequence of the other strand is complimentary because of the basepairing rules.

If you know the sequence of one strand, you can deduce the sequence of the complimentary strand

Ribonucleic acid (RNA): functions to express the genetic information present in DNA

RNA is similar to DNA but has 3 main differences.

1. Single stranded

2. Contains ribose rather than 2'-deoxyribose

3. The base uracil occurrs in place of thymine

Expression involves three types of RNA

1. Messenger RNA (mRNA)

Product of transcription

2. Ribosomal RNA (rRNA)

Combines with proteins to form ribosomes for translation of mRNA

3. Transfer RNA (tRNA)

Is charged with amino acids for protein synthesis during translation of mRNA

II. DNA Replication

See Figs. 3.5 A. and B.

Most genes of an organisms are arranged on chromosomes

Chromosomes

Most prokaryotic organisms (Bacteria) have one circular chromosome composed of DNA

The blueprint for a cell's structures and all biochemical processes

Bacterial growth = cell division

A growing (dividing) cell must replicate (make another copy) of its chromosome so that the two daughter cells will each receive a copy

 

Replication

Replication starts at the origin of replication, (ori), a specific site on the chromosome (or plasmid)

DNA polymerase: the enzyme that catalyzes the polymerization of deoxyribonucleotide triphosphates (dNTPs) during replication

Requires a short primer segment with a 3'-OH group to begin synthesis

 

Bi-directional: proceeds around the chromosome in both directions

Each new strand is synthesized in the 5' to 3' direction.

Original strand serves as the template for synthesis of a new complimentary strand

 

Semi-conservative

Each chromosome contains an original strand + newly synthesized strand

The set of 64 combinations of 3-base sequences (codons) present in genes.

4 different bases A, T (U), G, C can form 4³ = 64 different codons

E.g. 5' ATG ACG AAG AAC ATA ATT ATG ATC etc..........TGA 3'

(Note that codon tables usually use the transcribed sequence of the corresponding mRNA with U replacing T)

61 codons specify the 20 amino acids found in proteins

E.g. AAG encodes lysine (Lys)

ATG encodes methionine (Met)

The start codon --first codon of all genes

3 nonsense (stop) codons occur near the ends of genes and do not code for an amino acid

TAA, TAG and TGA

The code is degenerate (redundant). Most amino acids have more than one codon.

E.g. ACT, ACC, ACA and ACG all encode Thr

Codon bias --when one codon for an amino acid is used more often than the others

See Table 3.2

Different organisms have different preferences for redundant codons

Expression of a cloned gene from one species may be hindered in a different species.

This results when the tRNA with the anticodon for the rarely used codon is in short supply in the cell.

E.g. Expression of a human gene by a bacterium may not be efficient because the codon bias of bacteria and humans is different.

Structural gene: DNA segment that contains the codons specifying the sequence of amino acids of a protein. (Most genes are structural genes).

Also contains additional sequence information that affects its expression (E.g. promoter/operator, Shine-Delgarno sequence, transcription terminator)

See Fig. 3.10

Expression of a gene involves two main processes

1. Transcription

The transfer of genetic information from DNA to mRNA (the transcript)

Catalyzed by a RNA polymerase

2. Translation

Synthesis of a protein that has an amino acid sequence specified by the sequence of codons of the transcript

Eukaryotic genes contain noncoding segments of DNA (called introns) that interrupt the coding segments (called exons) of a gene.

Prior to translation, introns are removed from the primary transcript by mRNA processing to produce a functional transcript

See Fig. 3.11

 

The mRNA also has a G cap at the 5' end and a poly A tail at the 3' end

 

Other types of genes: Ribosomal genes encode rRNA and tRNA

Proteins, rRNA and tRNA are sometimes called gene products.

mRNA + ribosome + amino acid-tRNA -------> protein

See Fig. 3.14 for structure of amino acid-tRNA (charged tRNA)

Ribosome structure and composition in prokaryotes

1. Small subunit, 30S

16S rRNA + 21 proteins

2. Large subunit, 50S

5S rRNA + 23S rRNA + 32 proteins

Translation of mRNA

1. Initiation See Fig. 3.15

Shine-Dalgarno sequence (ribosome binding site) of mRNA binds to 30S ribosomal subunit via complementary base-pairing with a sequence of 16S rRNA

Anticodon of initiator Met-tRNA pairs with start codon of mRNA

(Eukaryotic transcripts do not have a Shine-Delgarno sequence)

2. Elongation See Fig. 3.17

1. Binding of another amino acid-tRNA by anticodon-codon pairing

2. Peptide bond formation between amino acids

Peptide bond (See Fig. 3.7)

3. Ejection of uncharged t-RNA

4. Translocation of mRNA and peptidyl-tRNA

3. Termination See Fig. 3.18

Occurs when a stop codon is encountered

Completed protein is release from the ribosome

Amino terminal end (First amino acid in protein, Met)

Carboxy terminal end (last amino acid)

Regulation of gene expression (Bacteria)

The genome of Escherichia coli K12 is about 4.7 million nucleotides long which encode about 4,500 genes

All of the genes are not expressed at the same time or at a constant rate

Constitutive genes encode proteins needed for growth and cell maintenance and are expressed constinuously

Expression is not regulated

Regulated genes encode proteins needed for growth and survival under variuos environmental conditions

The protein (gene product) is made only when needed by the cell

Ex. Genes that encode proteins involved in utilization of lactose are not expressed if glucose is available as a growth substrate

Allows cell to conserve vital resources (i. e. carbon and energy) by not making proteins that aren't needed

Regulation often occurs at the transcriptional level

Regulation of cloned gene expression is often employed in molecular biotechnology applications

Promoter is necessary for initiation of transcription.

See Fig. 3.19

DNA region upstream (towards the 5' end) of a gene.

Where RNA polymerase first binds to DNA for transcription of a gene

In E. coli, located 10 and 35 nucleotides upstream (positions -10 & -35) of the site of initiation of transcription (designated as +1)

Operator. DNA region near a promoter. Site were regulatory proteins bind to regulate the level of transcription.

Operon. A set of contiguous genes under the transcriptional control of a single promoter/operator.

Common in prokaryotes (e.g. lac operon has 1 promoter and 3 contiguous genes for utilization of lactose by cell)

In eukaryotes, each gene has its own promoter and regulatory sequences

I. Negative regulation.

Gene expression is affected at the transcriptional level by presence or absence of a repressor protein bound to the operator

Negative regulation is often exploited for applications in molecular biotechnology

II. Positive regulation

Transcription is increased by presence of an activator protein bound to the operator

Effector. Low molecular weight molecule that binds to a repressor or activator protein (e.g. glucose, amino acid)

Alters ability of a regulatory protein (repressor or activator) to bind to an operator

(Temperature may also be an effector)

Inducer: an effector that turns on gene expression

Corepressor: an effector that turns off gene expression

 

Repressor and activator regulatory proteins are encoded by genes called regulatory genes

III. Several other mechanisms for regulating gene expression exist in bacteria and higher organisms, but aren't often employed in molecular biotechnology

Two mechanisms with different outcomes

1. Induction of expression.

Effector binds to a repressor, prevents it from binding to the operator and the gene is transcribed

See Fig. 3.20

2. Repression of expression.

Effector (corepressor) binds to an inactive repressor, causes it to bind to the operator and transcription of the gene is blocked.

See Fig. 3.21

 

II. Positive regulation. Transcription is turned on or increased by an activator protein

Effector. Binds to an activator and affects its ability to bind to an operator.

As with negative control their are two possibilities (See Fig. 3.22)

Effector can:

1) Cause activator to bind to the operator and increase transcription

2) Prevent the activator from binding to the operator resulting in no or low level of transcription

Regulation of transcription in Eukaryotes

Mechanisms more complex than in prokaryotes

Terminology (See Fig. 3.23)

Transcription factor. Protein that regulates transcription

Response element. DNA sequence (also called a box) that is bound by a transcription factor

Enhancer. DNA sequence located at long distances from a gene that increases transcription

Eukaryotic genes are not arranged in operons.

Each gene has its own promoter and response elements.

Transcription involves formation of an initiation complex.

Composed of several transcription factors and RNA polymerase bound to the 5' end of a gene

(See Fig. 3.24)

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