Ch 14: Gene Expression (Transcription/Translation)

Transcription (def)

(genetics) the organic process whereby the DNA sequence in a gene is copied into mRNA (messenger RNA). The synthesis of RNA uses information in DNA molecule. DNA is used as a template to build a complementary RNA. The order of bases in the DNA will determine the order of bases on the RNA that is built. DNA is copied (transcribed) by messenger RNA (mRNA). This is also the first stage of gene expression. The base sequence of the DNA will determine the base sequence of the RNA. Transcription only transcribes one gene. After transcription is complete, the RNA leaves and the DNA double-helix comes back together. The three stages of transcription: - Initiation. - Elongation. - Termination.

Codons

*A codon is a group of 3 nucleotides. Three nucleotides will code for one amino acid.* Of the 64 triplets, 61 code for amino acids; 3 triplets are "stop" signals to end translation. The genetic code is redundant: more than one codon may specify a particular amino acid. But it is not ambiguous: no codon specifies more than on amino acid. Codons must be read in the correct *reading frame* (correct groupings) in order for the specified polypeptide to be produced. Codons are read one at a time in a non-overlapping fashion.

Nucleotide Mutations affect protein structure/function

*Mutations* are changes in the genetic material of a cell or virus. Changes to the Nucleotide sequence of DNA results in changes to the Amino Acid sequence of Polypeptides. *Point mutations* are chemical changes in just one or a few nucleotide pairs of a gene. The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein.

Initiation of transcription

*Promoters* (Nucleotide sequence upstream of the gene - It is a binding site for RNA Polymerase) signal the transcriptional start point and usually extend several dozen nucleotide pairs upstream of the start point. *Transcription factors* mediate the binding of RNA polymerase and the initiation of transcription. The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a *transcription initiation complex.* A promoter called a *TATA box* is crucial in forming the initiation complex in eukaryotes.

How many bases make up an anticodon?

3

Substitutions

A *nucleotide pair substitution* replaces one nucleotide and its partner with another pair or nucleotides. *Silent mutations* have no effect on the amino acid produced by a codon because of redundancy in the genetic code. *Missense muttaions* still code for an amino acid, but no the correct amino acid. Substitution mutations are usually missense mutations. *Nonsense mutations* change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.

Molecular components of translation

A cell translates an mRNA message into protein with the help of *transfer RNA (tRNA).* tRNAs transfer amino acids to the growing polypeptide in a ribosome. Translation is a complex process in terms of its biochemistry and mechanics.

inducer

A specific small molecule that binds to a bacterial repressor protein and changes the repressor's shape so that it cannot bind to an operator, thus switching an operon on.

Transcription: Elongation of the RNA strand

As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time. Transcription progresses at a rate of 40 nucleotides per second in eukaryotes. A gene can be transcribed simultaneously by several RNA polymerases. RNA transcript is built from 5' to 3' and Nucleotides are added to the 3′ end of the growing RNA molecule.

Inversions

Can result when changes occur in the orientation of chromosomal regions

Translation: Elongation of the Polypeptide chain

During elongation, amino acids are added one by one to the previous amino acid at the C terminus of the growing chain. Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation. Translation proceeds along the mRNA in a 5' to 3' direction.

Protein folding and post translational modifications

During synthesis, a polypeptide chain spontaneously coils and folds into its three dimensional shape. Proteins may also require post translational modifications before doing their jobs.

Structure and Function of Transfer RNA (tRNA)

Each tRNA translate a particular mRNA codon into a given amino acid. The tRNA contains an amino acid at one end and at the other end has a nucleotide triplet that can base-pair with the complementary codon on mRNA. tRNA molecules can base pair with themselves. Every tRNA has a group of three codons called the anticodon. tRNA *anticodons* base pair with an mRNA codon. Enzymes in the cell attach amino acids to tRNA. However, they only attach a specific amino acid to a specific tRNA (specific anticodon). (A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long).

Eukaryotic cells modify RNA after transcription

Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm. During RNA processing, both ends of the primary transcript are usually altered. Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

2 Steps for Accurate translation

First: a correct match between a tRNA and an amino acid, done by the enzyme *aminoacyl-tRNA synthetase.* Second: a correct match between the tRNA anticodon an an mRNA codon. Flexible pairing at the third base of a codon is called *wobble* and allows some tRNAs to bind to more than one codon.

Making polypeptides in bacteria and eukaryotes

In bacteria and eukaryotes multiple ribosomes translate an mRNA at the same time. Once a ribosome is far enough past the start codon, another ribosome can attach to the mRNA. Strings of ribosomes are called polyribosomes (or polysomes). Bacteria and eukaryotes can also transcribe multiple mRNAs from the same gene. In bacteria, the transcription and translation can take place simultaneously. In euks, the nuclear envelope separates transcription and translation.

operon

In bacteria, the region of bacterial DNA that regulates gene expression. Consists of four major parts: structural genes, regulatory gene, promotor gene, and operator.

Transfection

Insertion of recombinant DNA (plasmid) into animal cells.

Split Genes and RNA Splicing

Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions. These noncoding regions are called intervening sequences, or *introns.* The other regions are called *exons* because they are eventually expressed, usually translated into amino acid sequences. *RNA splicing* removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence. Many genes can give rise to two or more different polypeptides, depending on which segments are used as exons. This process is called *alternative RNA splicing.* RNA splicing is carried out by spliceosomes. *Spliceosomes* consist of proteins and small RNA's.

post translational regulation

Occurs when a cell has already made a protein, but it doesnt need to use the protein yet.

post transcriptional regulation

Occurs when the cell creates an RNA, but then decides that it should not be translated into a protein. RNAi molecules bind to an RNA via complementary base pairing. This creates double stranded RNA, which causes the RNA to be destroyed.

Types of Small-Scale (Point) Mutations

Point mutations within a gene can be divided into two general categories. - Nucleotide pair substitutions. - One or more nucleotide pair insertions or deletions.

Transcription & Translation in Prokaryotes vs Eukaryotes

Proks: - Transcription and Translation both happen in the cytoplasm. - Translation can actually begin before the RNA is completely finished being transcribed. Euks: - Transcription occurs in the nucleus, but translation occurs in the cytoplasm. - The RNA that results from transcription is unfinished, and will go through a process called RNA Processing before it is exported to the cytoplasm for translation.

Molecular components of Transcription

RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and joins together the RNA nucleotides. RNA polymerases assemble polynucleotides in the 5' to 3' direction. However, RNA polymerases can start a chain without a primer. The DNA sequence where RNA polymerase attaches is called the *promoter*; in bacteria, the sequence signaling the end of transcription is called the *terminator.* The stretch of DNA that is transcribed is called a *transcription unit.*

Ribozymes

Ribozymes are RNA molecules that function as enzymes. RNA splicing can occur without proteins, or even additional RNA molecules. The introns can catalyze their own splicing.

Mutagens

Spontaneous mutations can occur during DNA replication, recombination, or repair. *Mutagens* are physical or chemical agents that can cause mutations. Researchers have developed methods to test the mutagenic activity of chemicals. Most cancer causing chemical (carcinogens) are mutagenic, and the converse is also true.

Termination of Translation

Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome. The A site accepts a protein called a release factor, instead of tRNA. The release factor causes the addition of a water molecule instead of an amino acid. The release factor breaks the bond between the tRNA and the last amino acid; freeing the polypeptide, and the translation assembly comes apart. Translation is often no sufficient to make a functional protein. The large & small subunits disassociate.

Codons: Triplets of Nucleotides

The flow of info from gene to protein is based on a *triplet code:* a series of non-overlapping, three nucleotide words. The words of a gene are transcribed into complementary non-overlapping three nucleotide words of mRNA. These words are then translated into a chain of amino acids, forming a polypeptide. During transcription, one of the two DNA strands called the *template strand,* provides a template for ordering the sequence of complementary nucleotides in an RNA transcript. The template strand is always the same strand for any given gene. During translation, the mRNA base triplets, called codons, are read in the 5' to 3' direction. Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide.

Ribosome Association and Initiation of Translation

The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits. Translation occurs in at the ribosome. A small ribosomal subunit binds with mRNA and a special *initiator tRNA.* The initiator tRNA is the first tRNA to bind to mRNA and is responsible for recognizing the start codon. Then the small subunit moves along the mRNA until it reaches the start codon (AUG). The start codon establishes the reading frame for the mRNA. The addition of the large ribosomal subunit is last and completes the formation of the translation initiation complex. Proteins called initiation factors bring all these components together. Translation always starts with methionine.

Termination of Transcription

The mechanisms of termination are different in bacteria and eukaryotes. In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification. In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10-35 nucleotides past this polyadenylation sequence. *A termination sequence is just a nucleotide sequence that signals the end of transcription.*

Translation

The synthesis of a polypeptide using the genetic information encoded in an mRNA molecule. mRNA pairs to transfer RNA (tRNA) at ribosomes. *RNA is used as a template to build a polypeptide.* Ribosomes are the sites of translation. In prokaryotes, translation of mRNA can begin before transcription has finished. In eukaryotes, the nuclear envelope separates transcription from translation. Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA. Eukaryotic mRNA must be transported out of the nucleus to be translated. Genetic info flows from mRNA to protein through the process of translation. The base sequence of the RNA will determine the amino acid sequence of the polypeptide *3 Stages of translation: - Initiation. - Elongation. - Termination* All three stages require protein factors that aid in the process.

Ribosomes

These facilitate specific coupling of tRNA anticodons with mRNA codons during protein synthesis. The large and small ribosomal are made of proteins and *ribosomal RNAs (rRNAs).* In bacterial and eukaryotic ribosomes the large and small subunits join to form a ribosome only when attached to an mRNA molecule. A ribosome has three binding sites for tRNA: - The *P site* holds the tRNA that carries the growing polypeptide chain. - The *A site* holds the tRNA that carries the next amino acid to be added to the chain. - The *E site* is the exit site, where discharged tRNA's leave the ribosome.

RNA vs DNA structure

They both consist of nucleotide chains with the same basic building blocks (sugar, phosphate, nitrogenous base). However, DNA nucleotides are based on the sugar deoxyribose, while RNA nucleotides are based on the sugar ribose. This has consequences for the stability of the molecule. DNA is more stable. RNA is less stable, and thus is shorter-lived within the cell, and is more prone to mutations. DNA uses G, C, A, and T as the four nitrogenous bases. RNA uses Uracil in place of Thymine. DNA is typically double-stranded, though single-stranded DNA can and does exist, particularly in viruses. RNA is typically single-stranded, though double-stranded RNA can and does exist. tRNA is the most common example, but many viruses also utilize double-stranded RNA. RNA is the bridge between DNA and protein synthesis. Getting from DNA to protein requires two stages: transcription and translation. *Both are universal process amongst living things*

Targeting Polypeptides to Specific Locations

Two populations of ribosomes are evident in cells: free ribosomes (in cytosol) and bound ribosomes (attached to the ER). Free ribosomes mostly synthesize proteins that function in the cytosol. Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell. Polypeptide synthesis always begins in the cytosol. Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER. Polypeptides destined for the ER or for secretion are marked by a *signal peptide.* A *single recognition particle (SRP)* binds to the signal peptide. The SRP brings the signal peptide to its ribosome to the ER.

During RNA processing a(n) _____ is added to the 3' end and a(n)_____ is added to the 5' end of the RNA.

a long string of adenine nucleotides, modified guanine nucleotides.

Gene (def)

a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule. A gene is a stretch of DNA that serves as a blueprint for the construction of a polypeptide

insertions and deletions

additions or losses of nucleotide pairs in a gene. These mutations have a disastrous effect on the resulting protein more often than substitutions do. Insertion or deletion of nucleotides may alter the reading frame of the genetic message, producing a *frameshift mutation*

Duplications

can result in an extra copy of genes and are usually caused by unequal crossing over during meiosis or chromosome rearrangements. May result in new traits

Alteration of mRNA Ends

each end of a pre-mRNA molecule is modified in a particular way: the 5' end receives a modified nucleotide cap and the 3' end gets a poly-A tail. These modifications share several functions. - Facilitating the export of mRNA to the cytoplasm. - Protecting mRNA from hydrolytic enzymes. - Helping ribosomes attach to the 5' end.

RNA Processing (def)

mRNA modified before it is sent out of the nucleus (only occurs in eukaryotes). It prepares the RNA-transcript (comes directly from transcription) for use as mRNA. Prior to processing, the immature mRNA is referred to as pre-mRNA. Three major elements to RNA processing. 1) Addition of the 5' GTP cap. 2) Addition of the 3' Poly-Adenine tail. 3) Removal of introns. 5'GTP Cap: Protects the 5' end, prevents enzymatic degradation. 3' Poly-A tail: Protects the 3' end. Longer Poly-A tail a longer survival of the mRNA. Removal of Introns: introns are cut out, exons spliced together. *Introns:* mostly junk as they should not be translated. The function of introns is obscure and poorly understood. They were originally regarded as having no function but we now know that is often not the case. *Exons:* coding part of the mRNA and supposed to be used to make the protein.

translocation

occurs when part of one chromosome breaks off and attaches to another that causes the DNA sequence or gene to be lost, repeated, or interrupted

Transformation

process in which bacterial cells take up recombinant plasmid DNA/ giving bacteria foreign DNA

Primary transcript

the initial RNA transcript from any gene prior to processing.

Gene expression

the process by which DNA directs protein synthesis, includes two stages: transcription and translation