NOTES FOR BIOLOGY 1201

Section 001

Spring 2005

DR. STEVEN POMARICO

DNA is the instructions (program) that tells the cell what to do. Proteins are the results of those instructions.

Molecular Genetics: The Protein vs. DNA Debate - 6.1.1

>>>>>>The study of metabolic defects provided evidence that genes lead to proteins.

Archibald Garrod first proposed this relationship in 1909

         -suggested that genes dictated phenotypes via enzymes

         -inherited diseases were the result of the lack of an enzyme

>>>How genes control metabolism.

Continuing to Link Genes to Chemicals: Muller, Beable, and Tatum - 6.1.2

The suggestion of Garrod were confirmed in the 1930's

George Beadle and Edward Tatum conducted experiment with yeast, Neurospora crassa, and demonstrated that different mutants (generated by X-rays) had the pathway of arginine (an amino acid) synthesis blocked at different steps.

+ = growth, - = no growth

Wild-type yeast could survive on minimal medium. The mutants were auxotrophs that required added nutrients.

---Auxotrophs

Beadle and Tatum deduced that the 3 different mutant types each lacked a different enzyme in the pathway that synthesizes arginine.

From these results they formulated the one gene-one enzyme hypothesis

This has since been modified to the one gene-one polypeptide hypothesis because:

         -Not all proteins are enzymes

         -Many enzymes are comprised of 2 polypeptide subunits

>>>>>>The steps from DNA to protein.

Transcription and Translation: An Overview - 6.5.1

DNA ==> RNA ==> Proteins

>>>Step one - DNA to RNA

         This process is called transcription.

---Transcription

         -The RNA is complementary to the DNA

         -RNA that is synthesized from a gene coding for a protein is called messenger RNA

                    (mRNA)

DNA verses RNA

         -Similar because they are both polymers of nucleotides

         -Structural differences

-sugar - deoxyribose (DNA) verses ribose (RNA)

                   -base - thymine (DNA) is replaced by uracil (RNA)

>>>Step two - RNA to protein

         This process is called translation.

---Translation

Why translation??

         Different language of nucleic acids and proteins:

                   4 bases in nucleic acids

                   20 amino acids in proteins

Translation occurs on the ribosomes

In prokaryotes, since there is no nucleus, transcription and translation occur in rapid succession.

In eukaryotes the two processes are separated in time and space.

         -Transcription occurs in the nucleus

                   -The mRNA that is made must be modified before moving into the cytoplasm.

                             This modification process is called RNA processing

                                       and occurs only in eukaryotes.

         -Translation occurs in the cytoplasm.

>>>>>>Transcription of DNA to RNA

Transcription: RNA Formation from the DNA Template - 6.5.2

Like DNA replication, transcription of a DNA sequence to mRNA for a protein occurs by building the new molecule in a 5' => 3' direction. This means reading the template in the 3' => 5' direction.

During transcription of a gene, only one strand (templet strand) of the DNA’s two strands is read.

Different genes use different strands as the template strand.

The enzyme RNA polymerase catalyzes transcription.

         This enzyme:

                   1) Separates the DNA helix at a specific sequence

                                       The initiation sequence (a.k.a. startpoint)

                   2) Enzymatically synthesizes the RNA chain that is complementary to

                                       the 3' => 5' DNA strand

                   3) Stops the synthesis at the terminator

The initiation site + gene + terminator make up the transcription unit

---Transcription unit

>>>Initiation of transcription

RNA polymerase binds to the DNA at the promoter region of the gene.

---Promoter

In eukaryotes, RNA polymerase cannot recognize the promoter without the help of transcription factors

---Transcription factors

The transcription of eukaryote mRNA by RNA polymerase usually requires a specific transcription factor that binds to a DNA region known as a TATA box.

---TATA box

The RNA polymerase recognizes the TATA transcription factor-DNA complex and binds the DNA.

Other transcription factors or initiation factors may bind before transcription begins.

The active RNA polymerase separates the two DNA strands at the initiation site and transcription begins.

>>>Elongation of the RNA strand

Once transcription begins RNA polymerase performs two functions:

         1. Untwisting the DNA double helix for about ten nucleotides to expose the                    DNA template.

         2. Catalyze the linkage of new RNA nucleotide to the 3' end of the RNA polymer.

Elongation of mRNA occurs at about 30 - 60 nucleotides per second. As elongation proceeds:

                   -the RNA-DNA base pairs separates

                   -the DNA-DNA double helix reforms.

Several sequential RNA transcripts can be generated from a single gene. (i.e., as one transcript is being initiated others may be at various stages of elongation)

>>>Termination of transcription

Transcription: Termination and RNA Protection - 6.5.3

At the end of the DNA transcription unit for a gene there is a terminator.

---Terminator

         -May involve the interaction of termination factors with the DNA.

>>>>>>Eukaryotic cells modify RNA after transcription

         Before eukaryotic mRNA is exported from the nucleus it is processed in two ways:

                   1) Both ends are covalently altered

                   2) Intervening sequences (introns) are removed and the remaining                              sequence is spliced together

>>>Modification of the mRNA ends

During mRNA processing both the 5' and 3' ends are modified.

                   -The 5' end has a 5' cap added.

---5' cap

         -protects the mRNA from degradation

         -acts with the leader sequence to bind the mRNA to ribosomes.

                   -The 3' end of the mRNA has a poly-A tail added

---Poly-A tail

         -inhibits degradation

         -aids in export of mRNA from the nucleus

         -attached to a trailer sequence at the end of the mRNA

>>>RNA splicing

Posttranscriptional Modification / RNA Splicing - 6.5.4

In eukaryote the original RNA transcript is the complement of the DNA sequence for the gene, however the functional mRNA in the cytoplasm is much shorter.

Between transcription and translation the RNA is processed to remove parts of the sequence.

         -The original transcript is a precursor mRNA or pre-mRNA.

The DNA and the complementary pre-mRNA consists of the coding sequence interrupted by noncoding segments, called intervening sequences or introns.

---Introns

The coding sequences of the DNA and pre-mRNA are known as exons because they are the sequence that is expressed as proteins.

---Exons

The exon regions often code for different domains of a protein

---domains

Because these domains are on separate exons the opportunity for a function to be swapped from one protein to another is greater.

RNA splicing, as part of the RNA processing that occurs before the mRNA leaves the nucleus, removes the introns from the hnRNA

---RNA splicing

         -the boundary of the exons and introns is marked by sequences known as                    splicing sites.

         -RNA splicing also occurs during post-transcriptional modification of tRNA and             rRNA.

RNA splicing involves enzymes and other protein factors. In addition, small nuclear ribonucleoproteins (snRNPs) play a key role. Several snRNPs along with other proteins assemble to make a spliceosome.

---Spliceosome

The spliceosome brings together the exons and excises the intron. The exons are joined and the excised intron is released as a lariat-shaped loop.

Differential splicing (removal of all versus some introns) can result in to different mRNA products from a single pre-mRNA transcript.

>>>>>>Translation is the RNA-directed synthesis of polypeptides.

Translation: Ribosomal and Transfer RNA - 6.6.1

During translation, proteins are synthesized according to the genetic message of sequential codons in the mRNA.

---Codon

         -transfer RNA (tRNA) acts as the "interpreter" between the nucleotide “language”

                   of mRNA of the amino acid “language” of proteins.

         -In part of this role as “interpreter”, the tRNA must “read” the mRNA. This is

                   accomplished by the anticodon portion of the tRNA

---Anticodon

The Role of Transfer RNA: Charging a tRNA Molecule - 6.6.2

-The other portion the tRNA’s role as “interpreter” is to transfer the correct amino

                   acid from the cytoplasmic pool of amino acids to the ribosome for

                             protein synthesis. This is possible because each tRNA

                                       is specific for a single amino acid.

The specificity of this correct pairing is accomplished by a group of enzymes known as aminoacyl-tRNA synthetases

---Aminoacyl-tRNA synthetases

This is a two-step process

         1. Activation of the amino acid

         2. Transfer of the activated amino acid to the tRNA

This process occurs before the anticodon pairs up with the codon on the mRNA

Using this one codon => one anticodon => one amino acid method the gene is decoded to protein.

>>>The ribosome is where proteins are built.

The ribosome coordinates the pairing of tRNA anticodons with mRNA codons.

Eukaryotic ribosome structure

         -Two subunits (large and small)

         -Composed of 60% ribosomal RNA (rRNA) and 40% protein

         -The ribosomal subunits are made in the nucleolus.

         -The subunits combine as a ribosome only when they are translating a protein

In addition to the mRNA binding site (the groove between subunits), the ribosome also has 3 tRNA binding sites (P, A, and E)

The (peptidyl-tRNA binding) P site

         -holds the tRNA with the polypeptide chain attached.

The (aminoacyl-tRNA binding) A site

         -hold the aminoacyl-tRNA with the next amino acid to be added.

The (tRNA discharge or exit) E site

         -releases the tRNA once the peptide chain has been transferred to the next amino

acid

The ribosome holds all the components together as the next amino acid is transferred to the growing polypeptide chain.

>>>>>>In the genetic code, a triplet of nucleotides specifies an amino acid

Translation: Initiation Events - 6.6.3

Dictated by the math since there are 4 nucleotides and 20 amino acids.

         -if it was a 1:1 relationship then only 4 amino acids would be needed

         -a 2:1 would result in 16 possible amino acids (4²).

         -as a 3:1 there could be as many as 64 amino acids (4³)

So a triplet of nucleotides is the smallest size that could code for all the amino acids.

>>>The genetic "code"

61 of the 64 possible triplet code for amino acids. The remaining three triplets signal the translation to stop.

Since there are only 20 amino acids, more than one triplet can code for the same amino acid. This relationship is known as redundancy and usually the codons differ only at the third position.

There is no ambiguity in the triplet code since a given triplet codes for one and only one amino acid.

The correct ordering and grouping of nucleotide is an important aspect of the translation of the triplet codons. The correct ordering is the reading frame.

---Reading frame

The genetic “code” is nearly universal

         -There are few eukaryotic exceptions.

         -The genes in the mitochondria and the chloroplast can vary.

The first two positions of the anticodon compliment the codon exactly, the third position allows wobble in the base-pairing.

---Wobble

Because of wobble only 45 different tRNAs are needed to complement the 64 possible codons.

>>>Building a polypeptide.

Protein synthesis occurs in three stages:

                   1. Initiation

                   2. Elongation

                   3. Termination

Initiation of translation

         1. Assembly of mRNA, initiator tRNA (with methionine), and small ribosome               subunit.

                   Initiator tRNA binds to the mRNA start codon (AUG)

                   Requires protein initiation factors

         2. Large ribosome subunit joins complex, and the initiator tRNA is in the P site

                   The A site and E site are empty.

The elongation cycle of translation

Translation / Elongation: The Initiation of Elongation - 6.6.4

         1. The next tRNA occupies the A site and the anticodon hydrogen bonds to                the codon of the mRNA.

                   -energy for this step is provided by the hydrolysis of GTP=> GDP + P

                   -require a protein elongation factor

         2. Peptide bond formation

                   -the methionine from the initiator tRNA forms a peptide bond with the                              amino acid on the tRNA at the A site.

                   -this reaction as catalyzed by a ribozyme

                   -this leaves the tRNA at the P site with no amino acid, and the tRNA at                          the A site with a dipeptide attached.

         3. Translocation

                   -the “empty” tRNA moves to the E site and leaves

                   -the mRNA with the attached tRNA moves through ribosome in 5' => 3'                           direction. This translocates the tRNA (with the growing peptide)                                    from the A site to the P site

                   -energy for this step is provided by the hydrolysis of GTP=> GDP + P

                   -elongation proceeds at ~1 million amino acids per minute.

The termination of translation

Elongation Continued and Termination - 6.6.5

         1. The mRNA reaches a stop codon: UAA, UAG, or UGA

         2. A release factor binds to the A site

                   -there is no amino acid associated with the release factor.

         3. The peptidyl transferase adds H₂O instead of an amino acid to the end the              the polypeptide chain

                   -this frees polypeptide from tRNA in P site

>>>Polyribosomes can quickly make many copies of a protein from a single mRNA

Polypeptide Destinations: Signal Peptides and ER Ribosomes - 6.7.1

---Polyribosomes

         -Once a ribosome passes the initiation codon a new ribosome can bind and                    begin translation.

         -Several ribosomes may translate an mRNA at once, making many copies of                a polypeptide.

>>>Proteins are targeted for specific destinations by signal peptides

If a protein is to be inserted into a membrane (integral proteins) or to be shipped to a specific compartment (or out of the cell), then it is directed to the correct location by a signal peptide.

The signal peptide combines with a signal recognition particle (SRP) to direct the protein to the proper location.

---signal peptide

---signal recognition particle (SRP)

>>>Coupled transcription-translation in prokaryotes

In prokaryotes the mRNA may be translated as soon as the initiation codon is transcribed.

         -No nucleus to separate the new mRNA from the ribosomes.

         -No mRNA processing (splicing or modification) needed in prokaryotes

>>>>>>Point mutations can affect the function of a protein.

Genetic Mutation - 9.11.1

Genetic Mutation: Different Forms of Point Mutations - 9.11.2

---Point mutations

>>>Types of point mutations

                   A) Substitutions

---Substitutions

         -May have little or no effect

                   -Change may not change amino acids - wobble and redundancy

                   -Different amino acid may not cause a change.

         -Change may be drastic

Base-pair substitution mutations are usually missense mutations or nonsense mutations.

---Missense mutations

---Nonsense mutations

         B) Insertion or Deletion:

Genetic Mutation: Insertion and Deletion - 9.11.3

---Insertion

---Deletion

Both insertions and deletions can result in a frameshift mutation.

---Frameshift mutations

>>>Mutagenesis

Mutagenesis may be spontaneous or more often is the result of mutagens.

---Mutagens

Regardless of the cause of mutations the most common phenotypic effect is cancerous cell growth.