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From Gene to Protein

Gene expression

the process by which DNA directs the synthesis of proteins (or in some cases just the RNA) the expression of genes that code for proteins includes two stages: transcription and translation

DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins and of RNA molecules involved in protein synthesis

Evidence from study of metabolic defects

1909 Archibald garrod was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell
Biochemists since then have accumulated much evidence that cells synthesize and degrade most organic molecules via metabolic pathways and in which each chemical reaction in a sequence is catalyzed by a specific enzyme; such metabolic pathways lead, for instance, to the synthesis of pigments that give fruit flies their eye color

Garrod as the first to recognize that mendel's principles of heredity apply to humans as well as peas

Gene dictates the production of a specific enzyme

Beadle and Tatum

Hypothesis: Since Neurospora grows on minimal media, must have enzymes that convert simple compounds to amino acids necessary for growth

Neurospora species grow on a minimal medium containing sugar, inorganic salts and vitamin biotin
This means that they have metabolic pathways that makes proteins including enzymes
Neurospora species are all haploids
This means it is possible to study the expression of genes without worrying about dominance or recessive alleles.
Any mutations should be easy to detect since mutations will not be masked by another allele.
Irradiated a large number of Neurospora, and thereby produced some organisms with mutant genes
Mutation was linked to not being able to make the necessary amino acids
Tested all the amino acids, and found that the mutant grew with arginine
Some mutant spores could not grow without addition of specific amino acid---arginine
Arginine pathway is not a simple pathway, involves several different steps and several different enzymes
MOlecules are syntheized as a series of steps each catalyzed by an enzyme; see a final product only when every enzyme in the pathway is working properly.

Arginine Pathway

Starts with precursor (arg 1) to enzyme X; to produce ornithine...and so on
knowing the pathway, how do you know which enzyme is disrupted?

How do you know which enzyme is defective?

2nd: first gene inactivated, so enzyme A was damaged (protein damaged)

class II: enzyme B was damaged

Class III: mutant in gene c, didn't have enzyme C, so needed arginine
If you provide the substrate that bypasses the enzyme that converts it to substrate, you can know which enzyme is defective
Gene is damaged so protein wasn't made
GENE IS INVOLVED IN PROTEIN MAKING

Compound A-E are required to synthesize compound G necessary for growth, can you figure out the order in which they are synthesized by the wild-type cell?

1. The compounds that are used latest in the pathway will support the growth of the most mutants
2. The compounds that are used earliest in the pathway will allow the growth of the fewest mutants

Conclusions from Beadle and Tatum

Conclusions:
Genes control biochemical reactions by producing enzymes
Biochemical reactions in vivo (in living cells) consist of discrete, step by step reactions
Each reaction is specifically catalyzed by an enzyme
Each enzyme is specified by a gene
One gene one polypeptide idea

From gene to protein

DNA--->RNA (Transcription)
RNA--->Protein (translation)
Genes provide the instructions for making specific proteins; but a gene doesn't not build a protein directly. The bridge between DNA and protein synthesis is the nucleic acid RNA (had a ribose instead of deoxyribose and uracil not thymine)

Transcription

is the synthesis of RNA under the direction of DNA; both nucleic acids use the same language, and the information is simply transcribed, or copied, from one molecule to another
occurs in nucleus ; DNA serves as a template for assembling a complementary strand for RNA nucleotides

Messenger RNA

the copy of DNA made from the DNA template inside the nucleus; carries genetic message from the DNA to the protein-synthesizing machinery of the cell

Translation

synthesis of a polypeptide which occurs under the direction of mRNA; during this stage there is a change in language; the cell must translate the base sequence of mRNA into the amino acid sequence of a polypeptide; the sites of translation are ribosomes
Cytoplasm

Ribosomes

site of translation of amino acids to polypeptides; complex particles that facilitate the orderly linking of amino acids into polypeptide chains

BActeria vs Mammal

similar transcirption and translation;
But:
BActeria- lack of segregation between DNA and ribosomes allows transcription to still be going on when translation begins

Primary transcript

the initial RNA transcript form any gene, including those coding for RNA that is not translated into protein
(before it leaves for cytoplasm is is modified)

triplet code

when biologists began to suspect that the instructions for protein synthesis were encoded in DNA; there was a problem-only 4 nucleotide bases to specify 20 amino acids ; many bases must correspond to one amino acid

Flow of information from gene to protein is based on a triplet code

the genetic instructions for a polypeptide chain are written in DNA as a series of nonoverlapping, three nucleotide words; for example base triplet AGT at a particular position along a DNA strands results in placement of the amino acid serine at the corresponding position of the polypeptide being produced

Template Strand

only one of the two DNA strands is transcribed; this is the template strand because it provides the pattern or template for the sequence of nucleotides in an RNA transcript ; a given DNA strand is a template strand for some genes along a DNA molecule; for one gene-the same template is used every time

mRNA is complementary rather than identical to DNA template because RNA bases are assembled on the template according to base-pairing rules
RNA is syntheiszed in an anti-parallel strand

Codons

the mRNA base triplets to triplet DNA codes
During translation, the sequence of codons along an mRNA molecule is decoded, or translated, into a sequence of amino acids, making up a polypeptide chain; codons read in a 5'--->3' direction

ex. UGG is the codon for Trp;

Compare and contrast the functioning of DNA and RNA polymerases

How many codons are there?

64, all decoded by mid-1960s

The redundancy in the genetic code has a reason; the codons that specify the same amino acid only differ in the third base of the triplet

Termination codons

stop signals (UAA, UAG, UGA)
Do not designate amino acids

Reading frame

our ability to extract the intended message from a written language depends on reading the symbols in the correct groupings; read in non-overlapping codon triplets

In biology, a reading frame is a contiguous and non-overlapping set of three-nucleotide codons in DNA or RNA.
There are 3 possible reading frames in an mRNA strand and six in a double stranded DNA molecule due to the two strands from which transcription is possible.
An open reading frame starts with an atg (Met) in most species and ends with a stop codon (taa, tag or tga).

Evolution of genetic code

nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals

What polypeptide product would you expect from poly-G mRNA that is 30 nucleotides?

A polypeptide made up of 10 Gly (glycine) amino acids

The template strand of a gene contains the sequence 3' TTCAGTCGT'5'. Draw the nontemplate sequence and mRNA sequencd

Template: 3' TTCAGTCGT 5'
Nontemplate: 5' AAGTCAGCA 3'
mRNA sequence 5' AAGUCAGCA 3'

Imagine that the nontemplate sequence in question 2 was rascribed instead of the template sequence. Draw the mRNA sequence and translate it using figure 17.5. Predict how well the protein syntheiszed from nontemplate strand would function.

Template (from nontemplate sequence in problem writtein 3' to 5'):
3' ACGACTGAA 5'

mRNA sequence: 5' UGCUGACUU 3'
Translated: cys-stop-Leu

RNA Polymerase

pries the two strands of DNA apart adn joins the RNA nucleotides as they base-pair along the DNA template; like DNA polymerase that functions in DNA replication, RNA polymerases can assemble a polynucleotide only in its 5'--->3' direction; however RNA polymerases don't need a primer, can start a chain from scratch

promoter

the dna sequence where RNA polymerase attaches an initiates transcription is known as promoter; in bacteria, the sequence that signals the end of transcription is terminator
promoter is upstream from terminator

Start point of transcription, extends several dozen nucleotide pairs upstream from the start point, and determines which of the two strands of the DNA helix is used as the template

RNA polymerase in eukaryotes vs bacteria

Eukaryotes: 3 types
bacteria: 1 type

The coding region of a gene is 1500 base pair.How many amino acids exist in the protein encoded by this gene

500

An Average size protein is 400 amino acids.
How many nucleotides are required to code for the protein?

1200

transcription unit

the stretch of dna that is trascribed into RNA molecule

three stages of transcription

1. initiation=AFter RNA polymerase bind to the promoter, the DNA strands unwind, and the polymerase initaties RNA synthesis at the start point of the template strand
2. elongation= The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5' to 3'. In the wake of transcription, teh DNA strand re-form a double helix (orks on about 10 to 20 DNA bases at a time; adds nucleotides to the 3' end of the growing RNA molecule
3. termination of RNA chain=Eventually the RNA transcript is released, and the polymerase detaches from DNA; in bacteria transcription proceeds through a terminator sequence in the DNA; eukarytoes RNA polymerase II transcribes a sequence on the DNA called polyadenylation signal sequence

Transcription factors

a collection of proteins mediate the binding of RNA polymerase and the initiation of transcription ; proteins play a role in making the DNA more accessible to the transcription factors

Central Dogma

Cells are governed by a molecular chain of command with a directional flow of genetic information DNA -->RNA--->protein

Terminator

the sequence that signals the end of transcription

Transcription initiation complex

the whole complex of transcription factors and RNA polymerase II bound to the promoter

TATA Box

crucial promoter DNA sequence
Binding site for RNA polymerase

polyadenylation signal sequence

in termination stage of transcription; about 10 to 20 nucleotides down, proteins assocated with the growing RNA transcript cut it free fromt eh polymerase

Compare and contrast DNA polymerase and RNA polymerase in terms of function, template/primer, direction, and types of nucleotides

What is the promoter, and is it located at the upstream or downstream end of a transcription unit?

The region of DNA to which RNA polymerase binds to begin transcription; upstream end of the gene

What makes RNA polymerase start transcribing a gene at the right place ont he DNA in a bacteria cell? in a eukaryotic cell?

In a bacteria- RNA polymerase recognizes the gene's promoter and binds to it; Eukaryotic- transcription factors mediate the binding of RNA polymerase to the promoter

Before RNA leaves the nucleus, two things happen..

1. RNA transcript is modified
2. Non-coding regions are excised
Modification protects mRNA from being degraded in cytoplasm

RNA processing

both ends of the primary transcript are altered; certain interior sections of the RNA molecule are cut out and the remaining parts spliced together; these modifications produce a mRNA ready for translation

Alteration of mRNA ends

Each end of pre-mRNA molecule is modified in a particular way;
5' end modified first; receives a 5' cap modified form of a guanine nucleotide added onto the 5' end after transcription of the first 20 to 40 nucleotides

3' end of hte pre-mRNA molecules is also modified before the mRNA exist the nucleus; at teh 3' end, an enzyme adds 50 to 250 more adenine nucleotides forming a poly-A tail

5' cap

modified form of a guanine nucleotide added onto the 5' end

poly-A tail

at the 3' end, an enzyme adds 50 to 250 adenine nucleotides making this tail

Functions of poly-A tail and 5' cap

1. facilitate the export of the mature mRNA from the nucleus
2. they help protect the mRNA from degradation by hydroyltic enzymes
3. they help ribosomes attach to the 5' end of the mRNA once the mRNA reaches the cytoplasm

RNA splicing

The most remarkable stage of RNA processing occurs during the removal of a large portion of the RNA precursor molecule via splicing.

The average transcription unit is about 27,000 base pairs, but it only takes 1200 nucleotides to code for an average size protein of 400 amino acids; this means that most eukarytoic genes and their RNA transcripts have long noncoding stretches of nucleotides and sometimes noncoding stretches lie imbetween coding segments (make them split up)

RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

introns

noncoding segments of nucleic acid that lie between coding regions; intervening sequences

exons

eventually expressed; coding part; spliced together

the mRNA that enters the cytoplasm is an abridged version of what the RNA polymerase II originally produced

Ho s pre-mRNA spliced?

Particles called small nuclear ribonucleoproteins snRNPs recognize these splice sites;

snRNPs

located in the cell nucleus and are composed of RNA and protein molecules; RNA in snRNP is called small nuclear RNA snRNA
Several snRNPs join with additional proteins to make a larger assembly called a splicesome

splicesome

Several snRNPs join with additional proteins to make this larger assembly; interacts with certain sites along an intron, releasing the intron adn joining together the two exons that flanked the intron

What is in a splicesome?

1. pre-mRNA
2. proteins
3. small nuclear ribonucleoproteins (snRNPs)—ribozymes (catayltic activity)

snRNA base-pairs with nucleotides at the ends of the intron.
The RNA transcript is cut to release the intron, and the exons are joined together.

Ribozymes

RNA molecules that function as enzymes; idea of cataylitc role for snRNA arose from the discovery of these
Three properties enable some RNA molecules to function as enzymes
1. RNA is a single stranded, a region of an RNA molecule may base-pair with a complementary region elsewhere in the same molecule, which gives the molecule a particular 3-d shape
2. some of the bases RNA contain functional groups that may participate in catalysis
3. ability of rNA to hydrogen-bond with other nucleic acid molecules adds specificity to its catalytic activity

Functional importance of introns

The presence of introns increases the probability of potentially beneficial crossing over between genes. (exon shuffling)

There may also be occasional mixing and matching of exons between completely different genes.

One gene can encode more than one kind of a polypeptide

Proteins often have modular architecture consisting of discrete structural and functional regions called domains ; one domain of an enzymatic protein might include the active site, hile another might attach the protein to a cellular membrane

alternative RNA splicing

many genes are known to give rise to two or more different polypeptides, depending on which segments are treated as exons during RNA processing

domains

Proteins often have modular architecture consisting of discrete structural and functional regions

How does alteration of the 5' and 3' ends of pre-mRNA affect the mrRNA that exists int he nucleus?

The 5' cap and poly-A tail facilitate mRNA export from the nucleus, prevent the mRNA from being degraded by hydroyltic enzymes, and and facilitate ribosome attachment

How is RNA splicing similar to video editing

In editing a video introns are cut out and discarded, while exons are joined together so that the regions of joining are not noticeable

A gene that codes of ATPase has two alternatives for exon 4 and three alternatives for exon 7, how many different forms of a protein could be made from this gene?

6 different forms could be made because alternative splicing could generate six different mRNAs

Functional importance of poly-adenylation

1. Facilitate the export of mature RNA from nucleus to cytoplasm
2. Protects RNA from degradation by hydrolytic enzyme in cytoplasm
3. Help ribosome attachment to 5' end of the mRNA

What happens in translation?

a cell interprets a genetic message and builds a polypeptide accordingly ;

Message?

series of codons along a mRNA

interpreter?

Transfer RNA (tRNA)

tRNA

Function: to transfer amino acids from the cytoplasmic pool of amino acids to the ribosomes; cell keeps about 20 amino acids on stock
ribosome adds each amino acid brought to it by tRNA to the growing end of a polypeptide chain

molecules of tRNA are not the same; each type of tRNA translates a partciular mRNA codon into a particular amino acid

anticodon

at the other end of the tRNA is anucleotide triplet called an anticodon, which base-pairs with a complementary codon on mRNA

ex. mRNA has UUU (phenylalanine); tRNA base-pairs by hydrogen bonding AAA as its anticodon and carries phenylalanine at its other end;
As mRNA molecule moves through ribosome, phenylalnine will be added whenever the codon UUU is presented for translation

Where does amino acids attach?

3' end

Structure of tRNA

transcribed from DNA templates ; made in nucleus and mus ttravel from nucleus to cytoplasm; tRNA is used repeatedly

Single RNA strand that is only about 80 nucleotides long; because of the complementary stretches of bases that can hydrogen-bond to each other, this single strand can fold back upon itself adn form a molecule with a 3-d structure

twists and folds into a complicated 3'd shape

Accurate translation of a genetic message requires two processes that involve molecule recognition

1. tRNA that binds to an mRNa codon specifying a particular amino acid must carry that amino acid, and no other to the ribosomes
2. matching up the tRNA anticodon with the appropriate mRNA codon; if one tRNA variety exists for each mRNA codon that specifies an amino acid, there would be 61 tRNAs

Aminoacyl-tRNA synthetases

family of related enzymes; the correct matching up of tRNA and amino acid is carried out by aminoacyl-tRNA syntheases

Active site of this only fits a specific combination of amino acid and tRNA; 20 different synthetases, one for each amino acid ; the synthetases catalyzes the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP ; resulting is a tRNA or a charged tRNA, available to deliver its amino acid to a growing polypeptide chain on a ribosome

charged tRNA

available to deliver its amino acid to a growing polypeptide chain on a ribosome

Wobble:

flexible base pairing at htis codon position

Ribosomes

facilitate specific coupling of tRNA anticodons with mRNA codons during protein synthesis; a ribosome is made up of to subunits (large and small)

Ribosomal subunits-composed of?

proteins and RNA molecules called ribosomal RNAs or rRNAs; in eukaryotes made in nucleolus

Function of ribosomes

bringing mRNA and tRNA carrying amino acids together

has binding sites for mRNA and three for tRNA
P site, A site, E site

Catalyzes the formation of the peptide bond; as the polypeptide becomes longer, it passes through an exit tunnel in the ribosome's large subunit; when polypeptide is complete, it exits

P site

pepetidyl-tRNA site; holds the tRNA carrying the growing polypeptide chain

A site

holds the tRNA carrying the next amino acid to be added to the cain

E site

discharged tRNAs leave the ribosome from this site; exist site

Steps of translation

Translation can be divided into three stages:
Initiation
Elongation
Termination
All three phase require protein "factors" that aid in the translation process.
Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

Initiation

brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosome

1. small ribosomal subunits bind to a molecule of mRNA; mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon; an initator tRNA with the anticodon UAC, base-pairs with the start codon AUG; tRNA carries amino acid MET
2. The arrival of a large ribosomal subunit completes the initiation complex

Initiation factors

proteins required to bring all the translation components together ; GTP provides the energy

Elongation

peptide bonds between amino acids!

Termination

Termination occurs when one of the three stop codons reaches the A site.
A release factor binds to the stop codon
Hydrolyzes the bond between the polypeptide and its tRNA in the P site freeing the polypeptide
The translation complex disassembles.

Polyribosomes

Typically a single mRNA is used to make many copies of a polypeptide simultaneously.

When multiple ribosomes translate the same mRNA the entire unit is called a polyribosome or polysome

After translation

the process of translation is normally not enough to make a functional protein; modifications a polypeptide must undergo after translation

Protein folding

during synthesis, a polypeptide chain begins to coil and fold spontaneously due to amino acid sequence; forming a protein with a specific shape
Gene determines primary structure and primary structure determines shape

Chaperonin helps alot

post-translational modifications

may be required before the protein can begin doing its particular job in a cell; certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups or other additions
Enzymes may remove one or mroe amino acids from teh elading end of the polypeptide chain; sometimes polypeptide chains are enzymatically cleaved into to or more pieces
ex. Insulin polypeptide cut into two and held together by a disfuldie bond

free ribosomes

suspend and synthesize proteins to stay in cytoosol

bound ribosomes

make proteins of the endmembrane system to be secreted from the cell like insulin

single peptide

targets the protein in the ER; sequence of about 20 amino acids at or near the leading amino end of the polypeptide, is recognized as it emerges from the ribosome by a protein-RNA complex called a signal recognition particle

SRP

single recognition particle; functions as an adapter that brings the ribisome to a receptor protein built into the ER membrane ; this receptor is part of the a multiprotein translocation complex; polypeptide synthesis continues there adn the gorwing polypeptide snakes across the membrane into the ER lumen via a protein pore

Golgi apparatus

Chaperonin

Types of RNA

What two processes ensure that the correct amino acid is added to a growing polypeptide chain?

First, each aminoacyl0tRNA synthetase specifically recognizes a single amino acid and attaches it only to an appropriate tRNA

Second, a tRNA charged with its specific amino acid binds only to an mRNA codon for that amino acid

Describe how information of polyribosomes can benefit the cell?

Polyribosomes enable the cell to produce multiple copies of a polypeptide very quickly

Describe how a polypeptide to be secreted is transported to the endomembrane system?

A single peptide on the leading end of the polypeptide being synthesiezed is recognized by a signal-recognition particle that brings the ribosome to the ER membrane; there the ribosome attaches and continues to synthesize the polypeptide, depositing it on the ER lumen

Discuss the ways in which rRNA structure likely contributes to ribosomal function.

The structure and function of a ribosome depend more on rRNAs than on the ribosomal proteins

Mutations

responsible for the huge diversity of genes found among organisms because mutations are the ultimate source of new genes

point mutations

chemical changes in a single base pair of a gene

Point mutations has a negative genotypical expression

mutant condition is a genetic disorder or heriditary disease
ex. sickle clle can be traced to the change of a single nucleotide in the DNAs template strand makes an abnormal protein

Types of point mutations

base-pair substition
missense mutations
nonsense mutations
insertions
deletions
frameshift mutation
mutagens

base-pair substition

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

silent mutations

they have no effected on encoded protein (redundancy of the genetic code)

missense mutations

substitions that change one amino acid to another; have little effect on the protein; the new amino acid may have properties similiar to those of the amino acid it replaces or may be in the region of the protein where the exact sequence of amino acids isnt necessary for funciton

nonsense mutation

point mutation can also change a codon for an amino acid into a stop codon; it causes translation to be terminated early and the polypeptide will be shorter than the original ; lead to nonfunctioning proteins

Insertions/deletions

additions or losses of nucleotide pairs in a gene; these mutations have a disastrous effec ton resutling protein more often than substituions do;

May alter reading frame, the triplet grouping of bases on the mRNA that is read

Frameshit mutation

occur whenever the number of nucleotides inserted or deleted is not a multiple of three; changes reading frame

Mutagens

A number of physical and chemical agents, called mutagens , interact with DNA in ways that cause mutations.
In the 1920s, Hermann Muller discovered that X-rays caused genetic changes in fruit flies.
With X-rays, he was able to make Drosophila mutants that he could use in his genetic studies

What happens when one nucleotide pair is lost from the middle of the coding sequence of the gene?

In the mRNA, the reading frame downstream from the deletion is shifted, leading to a long string of incorrect amino acids in the polypeptide and, in most cases, a stop codon will arise, leading to premature termination; not functional!

What is the most important difference between bacteria and eukarytoes with regard to gene expression?

the bacteria's lack of compartmental organization

What is a gene

A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule

Would coupling of processes shwon in 17.24 be found in a eukaryotic cel?

No, because a eukartyoic cell is compartmentalized
Translation and transcription are separated in space and time in a eukartyotic cell due to compartments

In eukartyoic cells, transcription cannot begin until:

B several transcription factors have been bound to the promotoer

which of the following is not true of a codon?

D. it extends from one end of a tRNA molecule

The anticodon of a particular tRNA molecule is

A. complementary to the corresponding mRNA codon

Which of the following is not true of RNA processing?

A. Exons are cut out before mrRNA leaves the nucleus

True facts:
Nucleotides may be added at both ends of the RNA
Ribozymes may function in RNA splicing
RNA splicing can be catalyzed by splicesome
a primary transcript is often much longer than the final RNA molecule that leaves the nucleus

Using figure 17.5 identify a 5 to 3' sequence of nucletoides in the DNA tmplate strand for an mRNA coding for polypeptide sequence PH PRO LYS

. 5' CTTCGGGAA 3"

Which of the folloing mutations would be most likely to have a harmful affect on an organism

E. A single nucleotide insertion downstream of, and close to, the start codon

Whcih component is not directly involved in translation?

B. DNA

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