MOLECULAR GENETICS

I. THE CENTRAL DOGMA* *Coined by Francis Crick in 1958 ( A. Explains how genetic information moves around a cell and from parent to offspring under normal circumstances Replication (S.H.)

B. Exceptions to the Central Dogma 1. retroviruses have RNA, not DNA and then their RNA is used as a template to make DNA in the host cell a. Enzyme called reverse transcriptase catalyses the production of DNA from RNA

(points b and c are from b. can make DNA from mature mRNA (eg insulin) which can then be spliced into host's (eg bacteria) DNA without the introns. Then when the host's DNA is transcribed, proteins like insulin are made.

i. important that the DNA created by reverse transcriptase has no introns, because the host does not have the genes (and therefore proteins) necessary to remove the introns. 2. prions are misshapen proteins that have the ability to make normal proteins become misshapen as well and thus cause disease

Let's begin with a specific example of what we are trying to learn in this section:

II. Transcription A. the process of using a section of DNA (known as a gene) as a template to make a molecule of messenger RNA (mRNA) 1. each gene has the information for one polypeptide strand

B. only one strand of the two strands is transcribed i. Called the template strand or nonsense or anti-sense strand or non-coding strand C. the strand that is not transcribed is called the sense or coding or mRNA- like strand

D. only about 2% of the 3 billion base pairs in human DNA are part of genes 1. in total, there are about genes

E. Requirements 1. RNA polymerase a. An enzyme b. Many forms exist i. In eukaryotes usually a form called RNA polymerase II

2. Raw Materials a. 4 ribonucleoside triphosphates i. ATP/adenosine triphosphate ii. GTP/guanosine triphosphate iii. CTP/cytidine triphosphate iv. UTP/uridine triphosphate

F. Steps (in Prokaryotes) 1. Initiation a. RNA polymerase binds to the template strand of the DNA at an area called the promoter site i. by convention, the promoter is indicated on the sense/coding/ non-template strand

b. RNA polymerase opens the DNA molecule so that about 10 base pairs are separated 2. Elongation a. Complementary base pairing of free nucleoside triphosphates occurs with the exposed nitrogenous bases of the template strand

b. RNA polymerase binds the nucleosides to each other i. Two of the phosphates are released in the process c. The 5' end of a nucleoside is added to the 3' end of the RNA molecule being made

i. Means that the RNA is made in a 5' to 3' direction ii. Means that the template is being read in a 3' to 5' direction d. As the RNA polymerase moves along the DNA, it rewinds the DNA that has been transcribed and unwinds the DNA that has not yet been transcribed

3. Termination a. RNA polymerase reaches a termination site or terminator b. RNA polymerase releases the mRNA strand c. DNA rewinds completely

G. Note that more than one RNA polymerase can be transcribing one gene at the same time H. Other types of RNA are also made by transcription Here are 2 animations of transcription:

I. Transcription in Eukaryotes Here is an animation of transcription in prokaryotes and also in eukaryotes. Pay attention to the differences initiation requires proteins called transcription factors that RNA polymerase binds to 2. the promoter region has a sequence called the TATA box 3. the promoter often includes a CAAT sequence

4. product is called pre-mRNA and must be processed a. Synthesis of a cap made of a modified guanine attached to the 5' end i. protects the RNA from enzymes that work from the 5' end ii. Assembly point for the proteins needed to attract the small subunit of the ribosome to begin translation

b. introns (highly repetitive sequences that code for amino acids that are not found in the actual polypeptide) are removed i. Most begin with GU and end with AG c. what's left are sequences called exons (they are expressed)

i. joined together by spliceosomes (made of 5 types of snRNA and a large number of protein molecules) in a process called splicing d. Synthesis of a polyA tail, a stretch of adenine nucleotides attached to the 3' end

e. After processing mRNA can leave the nucleus Here is an animation of spliceosomes at work: *alternative and trans splicing makes it possible for a large number of proteins to be made from a small number of genes for more information see the following:

III. TRANSLATION A. the process of producing a polypeptide chain using the sequence of nitrogenous bases in mRNA as a template B. Requirements 1. Raw materials: amino acids

2. energy 3. ribosomes consisting of two subunits (small and large) a. Are separate when not in use for protein synthesis i. Small subunit consists of 1 rRNA molecule and 33 different protein molecules in eucaryotes

ii. Large subunit consists of 3 different rRNA molecules and about 45 different proteins in eucaryotes 4. mRNA with nitrogenous bases in triplets called codons 5. transfer RNA (tRNA) with a sequence of nitrogenous bases called an anticodon

a. Will complementary base pair with codons on the mRNA b. Each tRNA can bind to one amino acid

5. Wobble in anticodons a. Theoretically, there should be 64 types of tRNA's based on complementary base pairing with the mRNA codons b. Most organisms have between 31 and 40 tRNA's c. One type of tRNA will bind to more than one codon

d. Third base of a codon is allowed to bind inexactly (this position is known as the wobble position) i. G can bind with U in the wobble position ii. Inosine (a modified G) can pair with C, U or A in the wobble position

C. The Triplet Code 1. the mRNA nitrogenous bases are read in groups of three called codons 2. each codon calls for a particular amino acid

3. the code is degenerate meaning that more than one codon can code for the same amino acid a. there are 64 different codons possible but only 20 amino acids b. some codons are stop codons and signal the end of the polypeptide

D. Steps 1. Initiation a. Small subunit of ribosome binds to mRNA and moves along the mRNA until it reaches the start codon AUG b. Initiator tRNA binds at the P site on the large subunit

i. In eucaryotes, the start codon is AUG ii. Initiator tRNA has the anticodon UAC and carries the amino acid methionine c. Lastly, large subunit of ribosome binds

2. Elongation a. tRNA's that carry amino acids (charged tRNA's) diffuse to the A site on the ribosome b. If the anticodon base pairs are complementary to the codon base pairs, the tRNA will stay

c. A peptide bond is formed between the amino group of the incoming amino acid and the carboxyl group of the amino acid or peptide held at the P site d. The tRNA at the P site is released e. The growing polypeptide is on the tRNA located at the A site

f. The ribosome moves one codon forward from the 5' end to the 3' end i. The start codon is closer to the 5' end than is the stop codon g. The tRNA with the growing polypeptide is now at the P site and the A site is empty h. The steps from a to g repeat

3. Termination a. Ribosome's A site reaches a stop codon b. A release factor binds to the stop codon on the mRNA c. Polypeptide is released from the tRNA at the P site

d. Ribosome releases the mRNA and separates into 2 subunits

E. Polysomes occur when many ribosomes are translating the same MRNA

Here are 2 animations of translation and an interactive practice of trascription and translation: E. Fate of Molecules 1. mRNA: can be translated by other ribosomes or degraded and the nucleotides made available for reuse

2. tRNA: becomes charged again 3. ribosomes: available for reuse 4. polypeptide: a. Folds and coils into secondary and tertiary structures b. Associate with other polypeptides to form quaternary structures

c. transported to its final destination based on signal sequences (sequence of amino acids at terminal end) d. proteolysis: cutting polypeptide into smaller fragments e. glycosylation: addition of sugars f. phosphorylation: addition of phosphate groups

IV. Replication A. the synthesis of a new DNA molecule using an existing DNA molecule as a template B. semi-conservative because at end of replication, each new molecule is made of one old strand plus one new strand

C. Requirements 1. Raw materials a. 4 deoxyribonucleoside triphosphates i. deoxy- ATP/deoxyadenosine triphosphate ii. deoxy- GTP/deoxyguanosine triphosphate iii. deoxy-CTP/deoxycytidine triphosphate iv. deoxy- TTP/deoxythymidine triphosphate

2. helicase: an enzyme that unwinds the DNA double helix 3. DNA polymerase III: enzyme that a. binds to DNA and incoming nucleotides b. proofreads the growing new strand

c. can only add nucleotides to the 3' end of the growing chain d. Two work at the same time, one for each strand of DNA 3. RNA primase: enzyme that adds a short strand of RNA that complementary base pairs with each strand of DNA

a. initiates DNA replication by producing a 3' end that DNA polymerase can add nucleotides to 4. DNA ligase: links together short chains of DNA called Okazaki fragments

5. DNA Polymerase I: enzyme that removes RNA primers and replaces them with DNA a. can only add nucleotides to the 3' end of a nucleotide chain

6. telomerase: enzyme that replaces telomeres a. telomeres: region of DNA at the ends of chromosomes that help to prevent against DNA erosion b. telomerase mostly found in immortal cells and in the developing organism

D. Process 1. helicase unwinds the double helix a. becomes unstable b. open area is called a replication bubble or fork

2. single-strand binding protein prevents the two strands from re- joining The following diagram and all similar ones are from:

3. RNA primase puts a short strand of RNA that is complementary to each strand of the separated DNA a. needed because the DNA polymerase need a 3' end to add nucleotides to

3. RNA primase puts a short strand of RNA that is complementary to each strand of the separated DNA a. needed because the DNA polymerase needs a 3' end to add nucleotides to

3. RNA primase puts a short strand of RNA that is complementary to each strand of the separated DNA a. needed because the DNA polymerase needs a 3' end to add nucleotides to

b. Only 1 primer is needed for the DNA strand that ends with the 3' end (leading strand) i. because the primer will end with a 3' end c. Problem with the strand that ends with the 5' end (lagging strand) is that the primer will end with the 5' end

i. DNA polymerase cannot add nucleotides to the 5' end ii. Must make short strands of DNA on the 3' end of the primer iii. results in short strands of DNA called Okazaki fragments 4. DNA sequence is now copied but the new nucleic acid is in short pieces and has many bits of RNA

5. DNA polymerase I finds the places where an RNA primer and a short strand of DNA are not joined together a. Begins from the 5' end and removes the RNA primer b. Leaves a 3' end at the end of the DNA free c. Adds nucleotides to the free 3' end

6. the strands are still in bits 7. ligase joins all the DNA bits together

8. Now there are two new double- stranded molecules of DNA

9. in eucaryotes, replication occurs simultaneously at many sites on the DNA molecule m

Here are animations of the replication process: (there are two parts here) V. Mutations A. mutation: heritable changes in DNA 1. somatic mutations are only passed on to daughter cells