bio211 chapter 17 notes

what is gene expression?

The process by which DNA directs the synthesis of proteins

What was Beadle and Tatum’s experiment?

1. Individual neurospora cells were placed on a complete medium

2. The cells were subjected to X-rays to induce a mutation in one gene per cell

3. Each surviving cell formed a colony of genetically identical cells

4. Cells from each colony were placed in a vial with minimal medium. Cells that did not grow were identified as nutritional mutants

5. Cells from one nutritional mutant colony were placed in a series of vials, plus one additional nutrient (valine, lysine, arginine)

What is the one gene-one enzyme hypothesis?

A gene correlates the production of specific enzyme function

What is transcription?

The synthesis of RNA using information in DNA→ the two nucleic acids are written in different forms of the same language

What is mRNA?

Messenger RNA→ carries genetic messages from DNA to the protein-synthesizing machinery of the cell

What is translation?

Synthesis of a polypeptide using hte information in mRNA→ cell must translate the nucleotide sequence of an mRNA molecule into the amino acid sequence of a polypeptide

What is the site of translation?

Ribosomes

What is the difference between transcription/translation in bacteria and eukaryotic cells?

1. In bacteria, transcription/translation occur simultaneously as there is no nucleus to separate the processes

2. In eukaryotic cells, transcription/translation occur in steps, as there is a nucleus to delay the translation

What is a primary transcript?

The initial RNA transcript from any gene that is not translated into protein

What is the triplet code?

The genetic instructions for a polypeptide chain are written in DNA as as series of nonoverlapping, three-nucleotide words

What is the template strand?

The DNA strand that is used to provides pattern for the sequence of nucleotides in an RNA transcript

How is an mRNA molecule synthesized?

synthesized as complementary to the template strand→ antiparallel

If template strand is in 3’ to 5’ direction, the RNA molecule will be synthesizeddin 5’ to 3’ direction

What are codons?

mRNA nucleotide triplets written in 5’ to 3’ direction

What is the coding strand?

no template DNA strand→ sequence of coding strand is identical to mRNA (except T for U)

What this the RNA polymerase?

Enzyme that ttprties two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand

• assemble only in 5’ too 3’ direction

• can start chain from scratch→ do not need to add the first nucleotide onto a pre-existing primer

What are the 3 stages of transcription?

1. initiation

2. elongation

3. termination

What is the process of initiation?

After RNA polymerase binds to the promoter, the polymerase unwinds the DNA strands and initiates RNA synthesis at the start point on the template strand

What is the process of elongation?

The RNA polymerase moves downstream, unwinding the DNNA and elongating the RNA transcript in a 5’ to 3’ direction

• After the transcription has occurred, the DNA strands re-form a double helix

What is the process of termination?

After the RNA transcript is released, the polymerase detaches from the DNA

What is the promoter?

DNA sequence that the RNA polymerase attaches to and initiates transcription

What is the terminator?

Sequence that signals the end of transcription

What is a transcription unit?

Stretch of DNA downstream that from the promotor that is transcribed into an RNA molecule

What is the start point?

The promoter sequence of a gene→ RNA polymerase binds in a precise location and orientation on the promoter

What is the process of initiation of transcription at a eukaryotic promoter?

1. A TATA box (nucleotide sequence containing TATA) about 25 nucleotides upstream from the transcription start point

2. Several transcription factors will work, with one recognizing the TATA box, must bind to the DNA before the RNA polymerase 2 can bind in the correction position and orientation

3. Additional transcription factors bind to the DNA along the RNA polymerase 2, forming the transcription initiation complex

What are transcription factors?

Collection of proteins in eukaryotic cells that help guide the binding of RNA polymerase and the initiation of transcription

• only after the transcription factors are attached to the promotor does RNA polymerase 2 attach as well

What is the process of termination of transcription in eukaryotic cells?

RNA polymerase 2 transcribes a sequence on the DNA→ polyadenylation signal sequence (AAUAAA) in the pre-mRNA

• once this sequence appears, about 10-35 nucleotides down from the sequence, theses proteins cut the RNA transcript free from the polymerase, releasing hte pre-mRNA

What is RNA processing?

Both ends of the primary transcript are altered, and interior sections of the RNA molecule are cut out and the remaining parts are spliced together

What is the process of alteration of mRNA ends?

1. the 5’ end is synthesized first, receiving a 5’ cap (modified form of Guanine nucleotide) added onto the 5’ end after transcription of the first 20-40 nucleotides have been transcribed

2. An enzyme adds 50-250 more adenine (A) nucleotides at the 3’ end→ poly-A tail

What is the purpose of the 5’ cap and poly-A tail?

1. Facilitate the export of mature mRNA from the nucleus

2. Protect the mRNA from degradation by hydrolytic enzymes

3. help ribosomes attach to the 5’ end of the mRNA once it reaches the cytoplasm

What is RNA splicing?

Large portions of the RNA primary transcript molecule are removed and the remaining portions are reconnected

What are introns?/

non-coding segments of nucleic acid that lie between coding regions (intervening sequences)

What are exons?

Expressed regions of nucleotides

• UTRs of the exons at the end of RNA are not translated into protein

What is a spliceosome?

Large complex made of proteins and small RNAs

• binds to several short nucleotide sequences along an intron, including key sequences at each end

What is the process of spliceosome splicing?

1. complex binds to serveral short nucleotide sequences along an intron, including key sequences at each end

2. Intron is released (rapidly degraded), and the spliceosome joins together the two exons that flanked the intron

3. The small RNAs in the spliceosome also catalyze the splicing reaction

*RNAs can act as catalysts

What are ribozymes?

RNA molecuels that function as enzymes

• intron RNA functions as a ribosome and catalyzes its own removal

What 3 properties allows RNA molecules to function as enzymes?

1. RNA is single-stranded→ a region of an RNA molecule may base-pair, in an antiparallel arrangement with a complementary region elsewhere in the same molecule

• 3D structure for catalytic function (ribozymes)

2. Some of the bases in RNA contain functional groups that can participate in catalysis

3. The ability of RNA to hydrogen-bond with other nucleic acid molecules (RNA/DNA) adds specificity to its catalytic activity

What is an example of RNA’s H-bond specificity?

Complementary base pairing between the RNA of the spliceosome and the RNA of a primaryRNA transcript precisely locates the region where the ribozyme catalyzes splicing

What is alternative RNA splicing?

Different genes can give rise to 2+ different polypeptides

• dependent on which segments are treated as exons during RNA processing

What are domains?

discrete structural and functional regions

• one domain of an enzyme could include an active site, while another domain could allow the enzyme to bind to a cellular membrane

What is exon shuffling?

Introns increase the probabilities of crossing over between exons of alleles of a gene

• provides more terrain for crossovers without interrupting coding sequences

• Occasional mixing and matching of exons between completely different (nonallelic) genes

What is a tRNA and its properties?

Transfer RNA

• transferes an amino acid from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome

• Single RNA strand that is only 80 nucleotides long→ complementary base pairing between nucleotides allows for 3D structure

What is the anticodon?

Particular nucleotide triplet that base-pairs to a specific mRNA codon

What shape is a tRNA?

2D structure of a clover

What are the properties of anticodons?

Written 3’ to 5’ to align with codons written as 5’ to 3’

• base-pairs with codons by hydrogen bonding

Where is tRNA made from?

Made in nucleus

What is the process of tRNA translation?

1. TRNA binds to an mRNA codon specifying a particular amino acid must carry that amino acid to the ribosome

2. The correct matching of tRNA and amino acid is carried out by aminoacyl-tRNA synthetases

3. The active site of each aminoacyl-tRNA synthetase fits only a specific combination of amino acid/tRNA

How many synthetases are there?

20→ matches with 20 amino acids

What are the properties of synthetases?

1. A synthetase joins a given amino acid to an appropriate tRNA

2. One synthetase is able to bind to all of the different tRNAs for a specific amino acid

3. Catalyzes the covalent attachment of amino acid to its tRNA in a process driven by hydrolysis of ATP→ results in aminoacyl tRNA

• released from enzyme and is then available to deliver its amino acid to a growing polypeptide chain on a ribosome

What is a wobble?

A flexible base pairing at the 5’ to 3’ codon position→ explains why synonymous codons for a given amino acid most often differ in their third nucleotide base

What else has properties of a wobble?

a tRNA with the antcodon 3’ UCU 5’ can base pairing between nucleotides with either mRNA codon 5’ AGA 3’ or 5’ AGG 3’

what are rRNAs?

Ribosomal RNAs→ subunits made in the nucleolus in eukaryotes

What is the most abundant type of RNA?

rRNA

What is an example of ribosomal complex?

1. the ribosome has a binding site for mRNA as well as three binding sites for tRNA→ P, A, E site

2. the P site holds the tRNA carrying the growing polypeptide chain

3. A site holds the tRNA carrying the next amino acid to the next chain

4. Discharged tRNAs leave the ribosome from the E site

5. The ribosome holds tRNA and mRNA in close proximity and positions→ new amino acid can be added to carboxyl end of growing polypeptide chain

6. It then catalyzes the formation of the peptide bond→ polypeptide becomes longer, passing through exit tunnel

What are the 3 stages of polypeptide synthesis?

1. initiation

2. elongation

3. termination

What are the steps of the initiation stage?

Brings together an mRNA, tRNA w/ first amino acid of polypeptide, and two subunits of a ribosome

1. A small ribosomal subunit binds to both mRNA and a specific initiator tRNA, which carries methionine

2. In eukaryotes, the small subunit w/ the initiator tRNA already bound, binds to the 5’ cap of the mRNA until it reaches the AUG start codon→ initiator tRNA then h-bonds

• AUG is extremely important→ establishes the codon reading frame for the mRNA

What is the translation initiation complex?

union of mRNA, initiator tRNA, and small ribosomal subunit w/ attachment of a large ribosomal subunit

What is N-terminus?

initial methionine at the amino end

What is the C-terminus?

Final amino acid at carboxyl end

What is the process of the elongation cycle of translation?

1. Codon recognition→ anticodon of an incoming aminoacyl tRNA base-pairs w/ the complementary mRNA codon in the A site

• hydrolysis of GTP inc the accuracy/efficiency of this step

2. Peptide bond formation→ an rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between the carboxyl end of the growing polypeptide in the P site and the amino group of the new amino acid in the A site

3. Translocation→ The ribosome translocates the tRNA in the A site to the P site→ the empty tRNA in the P site is moved to the E site, where it’s released. MRNA moves along with its bound tRNAs, brining the next codon to be translated into the A site

How do you read a triplet codon on a codon table?

1st nucleotide→ horizontal category

2nd nucleotide→ vertical category

3rd nucleotide→ category within box

What is the process of termination in translation?

1. When a ribosome reaches a stop codon on an mRNA, the A site of the ribosome accepts a “release factor”, a protein shaped like a tRNA instead of an aminoacyl tRNA

2. The release factor promotes hydrolysis of the bond between tRNA in the P site and the last amino acid of the polypeptide, thus freeing the polypeptide from the ribosome

3. The two ribosomal subunits and the other components of the assembly dissociate w/ the hydrolysis of two GTP molecules

What is a signal peptide?

Peptide which targets protein to the Endoplasmic reticulum (ER)

• found near the leading end (N-terminus) of the polypeptide is recognized as it emerges by a signal-recognition particle (SRP)

What is the process of the signaling mechanism for targeting proteins to the ER?

1. Polypeptide synthesis begins on a free ribosome in the cytosol

2. An SRP binds to the signal peptide, halting synthesis momentarily

3. The SRP binds to a receptor protein in the ER membrane, part of a protein complex that forms a pore

4. The SRP leaves, and the polypeptide synthesis resumes, with simultaneous translocation across the membrane

5. The signal peptide is cleaved by an enzyme in the receptor protein complex

6. The rest of the completed polypeptide leaves the ribosome and folds into its final conformation

What are polyribosomes (polysomes)?

Strings of ribosomes that are found to trail along the mRNA after the riboosome is far enough past the start codon

What are mutations?

Changes to the genetic info of the cell

What are point mutations?

Changes in a single nucleotide pair of a gene

what is a nucleotide-pair substitution?

Replacement of one nucleotide and its partner with another pair of nucleotides

What is a silent mutation?

A mutation with no observable effect on phenotype

What are missense mutations?

Substitutions that change one amino acid to another one

What is a nonsense mutation?

Causes a translation to be terminated prematurely and hte resulting polypeptide witll be shorter than the original one encoded by a normal gene

What are insertions and deletions?

Additions or losses of nucleotide pairs in a gene→ have a disastrous effect on the resulting protein more often than substitutions

What is a frame shift mutation?

A mutation thatt occurs whenever the number of nucleotides inserted/deleted is not a multiple of 3→ all nucleotides downstream will be improperly grouped

What are mutagens?

Physical and chemical agents that can alter DNA

What is CRISPR-Cas9?

Cas 9→ nuclease that cuts double-stranded DNA molecules

• The CAs 9 protein will cut any sequence to which it is directed

What is the process of gene editing using CRISPR-Cas 9?

1. Cas9 protein and guide RNA are allowed to bind to each other, forming a complex that is then introduced into a cell

2. In the nucleus, the complementary sequence of the guide RNA binds to the part of the target gene. The active sites of the Cas9 protein cut the DNA on both strands

3. The broken strands of DNA are “repaired” by the cell in one of two ways

• disabling the target gene to study its normal function

• If target gene has a mutation, it can be repaired by providing a normal copy of the gene