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Gene Expression: From Gene to Protein (((((((((Mutations, mutations are…
Gene Expression: From Gene to Protein
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Translation and Transcription cont.
Basic Principles of Transcription and Translation
genes provide the instructions for making specific proteins
the bridge between DNA and protein synthesis is the nucleic acid RNA
RNA is chemically similar to DNA except that it contains ribose instead of dioxyribose as its sugar and has the nitrogenous base uracil rather than thymine
so RNA strands have AUCG (A+U and C+G) and usually only consist of a single strand
both nucleic acids and proteins are polymers with specific sequences of monomers that convey information
in DNA or RNA, the monomers are the four types of nucleotides
genes are usually hundreds to thousands of nucleotides long, each gene having a specific sequence of nucleotides
getting from DNA to protein requires two major stages: transcription and translation
Transcription and Translation
transcription
is the synthesis of RNA using information in the DNA
the two nucleic acids are written in different forms of the same language, and the information is simply transcribed (or rewritten) from DNA to RNA
for a protein-coding gene, the resulting RNA molecule is a faithful transcript of the gene's protein-building instructions
this type of RNA molecule is called
messenger RNA (mRNA)
bc it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell
translation
is the synthesis of a polypeptide using the information in the mRNA
the cell must translate the nucleotide sequence of an mRNA molecule into the amino acid sequence of a poly peptide
^ the sites of translation are
ribosomes
(molecular complexes that facilitate the orderly linking of amino acids into polypeptide chains
transcription and translation happen in all organisms
Nutritional Mutants in Neurospora
a breakthrough came a few years later where Beadle and Edward Tatum began working with
Neurospora crassa
, a bread mold and a haploid species
they bombarded the mold with X-rays (bc they're known to cause mutations) and looked among the survivors for mutants that differed in their nutritional needs from the wild-type bread mold
it can grow on a simple solution containing minimal nutrients for growth - inorganic salts, glucose, and the vitamin biotin - incorporated into agar
they generated different "nutritional mutants" of
Neurospora
cells, each of which was unable to synthesize a particular essential nutrient
such cells could not grow on minimal medium but could grow on complete medium but could grow on a complete medium, which contains all nutrients needed for growth
for a complete medium, consists of the minimal medium supplemented with all 20 amino acids and a few other nutrients
Beadle and Tatum hypothesized that in each nutritional mutant, the gene for the enzyme that synthesizes a particular nutrient had been disabled
Genes specify proteins via transcription and translation
in 1902, British physician Archibald Garrod was the first to suggest that gene dictate phenotypes through enzymes (proteins that catalyze specific chemical reactions in the cell)
he said that the symptoms of an inherited disease reflect the inability to make a particular enzyme
^he later referred to such diseases as "inborn errors of metabolism"
years later, evidence supported Garrod's hypothesis that a gene dictates the production of a specific enzyme, later named the
one gene-one enzyme hypothesis
biochemists learned that cells synthesize and degrade most organic molecules via metabolic pathways (in which each chemical reaction in a sequence is catalyzed by a specific enzyme)
What is gene expression anyway?
is the process by which DNA directs the synthesis of proteins (or in some cases, just RNAs)
the expression of genes that code for proteins includes two stages: transcription and translation
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Cracking the Code
the first codon was deciphered in 1961 by Marshell Nirenberg
he synthesized an artificial mRNA by linking together many identical RNA nucleotides containing uracil as their base
no matter where the genetic message started/stopped, it could contain only one codon (UUU) over and over
he then added this "poly-U" polynucleotide to a test tube mixture containing amino acids, ribosomes, and the other components required for protein synthesis
his artificial system translated the poly-U mRNA into a polypeptide containing many units of the amino acid phenlalanine (Phe or F)
he then determined that the mRNA codon UUU specifies the amino acid phenlalanine
all 64 codons were deciphered by the mid-1960s
61 of the 64 triplets code for amino acids
the three codons that do not designate amino acids are "stop" signals (termination codons) marking the end of translation
The Genetic Code
there's only four nucleotide bases to specify 20 amino acids
how many nucleotides, then, would turn out to correspond to an amino acid?
experiment shave shown that the flow of information from gene to protein is based on a
triplet code
: the genetic instructions for a polypeptide chain are written in the DNA as a series of nonoverlaping, three-nucleotide words
during transcription, the gene determines the sequence of nucleotide bases along the length of the RNA molecule that is being synthesized
for each gene, only one of the two strands of DNA is transcribed
^ this strand is called the
template strand
bc it provides the pattern for the sequence of nucleotides in an RNA transcript
RNA nucleotides are assembled on the template according to base-pairing rules
these pairs are similar to the ones that form during DNA replication, except that uracil (U) pairs with A and the mRNA nucleotides contain ribose instead of deoxyribose
the RNA molecule is synthesized in an antiparallel direction to the template strand
the mRNA nucleotide triplets are called
codons
(usually written in the 5' to 3' direction)
EX: UGG (the codon that translates into the amino acid tryptophan )
codon is also used for the DNA nucleotide triplets along the nontemplate strand. these codons are complementary to the template strand and identical in sequence to the mRNA (except they have a T wherever there is a U in the mRNA)
for this reason ^, the nontemplate DNA strand is often called the
coding strand
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Split Genes and RNA Splicing
RNA splicing - where large portions of the RNA molecules are removed and the remaining portions are reconnected
the average length of a transcription unit along a human DNA molecule is about 27,000 nucleotide pairs (the RNA transcript is also this long)
each amino acid is coded by a triplet of nucleotides
the average size protein of 400 amino acids requires only 1,200 nucleotides in RNA to code for it
the noncoding segments of nucleic acid that lie between coding regions are called
i
ntervening sequences, or
introns
the other regions are called
exons
, because they are eventually
e
xpressed, usually by being translated into amino acid sequences
in RNA splicing, the introns are cut out from the molecule and exons joined together, forming an mRNA molecule with a continuous coding sequence
How is pre-mRNA splicing carried out?
the removal of introns is accomplished by a large complex made of proteins and small RNAs called a
spliceosome
this complex ^ binds to several short nucleotide sequences along an intron
the intron is then released, rapidly degraded, and the spliceosome joins together the two exons that flanked the intron
small RNAs in the spliceosome participate in spliceosome assembly and splice sit recognition and catalyze the splicing reaction
Eukaryotic cells modify RNA after transcription
during
RNA processing
, both ends of the primary transcript are altered
also, in most cases, certain interior sections of the RNA molecule are cut out and the remaining parts spliced together
these modifications produce an mRNA molecule ready for translation
Alteration of mRNA Ends
the 5' end of a pre-mRNA molecule (which is synthesized first) receives a 5' cap, a modified form of a guanine (G) nucleotide added onto the 5' end after transcription of the first 20-40 nucleotides
the 3' end is also is modified before the mRNA exits the nucleus
at the 3' end, an enzyme adds 50-250 more adenine (A) nucleotides, forming a
poly-A tail
the 5' cap and poly_A tail share several functions:
they facilitate the export of mature mRNA from the nucleus
they help protect the mRNA from degradation by hydrolytic enzymes
they help ribosomes attach to hte 5' end of the mRNA once the mRNA reaches the cytoplasm
Mutations
mutations are responsible for the huge diversity of genes found among organisms
mutations are the ultimate source of new genes
point mutation cause changes in a single nucleotide pair of a gene
if a point mutation occurs in a gamete or in a cell that gives rise to gametes it may be transmitted to offspring and to future generations
small-scale mutations within a gene can be divided into 2 categories
:
single nucleotide-pair substitutions
nucleotide-pair insertions or deletions
Substitutions
a nucleotide-pair substitution is the replacement of one nucleotide and its partner with another pair of nucleotides
some substitutions have no effect on the encoded protein
silent mutation
- has no observable effect on the phenotype (can also occur outside of genes)
missense mutation
- substitutions that change one amino acid into another one
nonsense mutation
- when a point mutation can also change a codon for an amino acid into a stop codon
it causes translation to be terminated prematurely; the resulting polypeptide will be shorter than the polypeptide encoded by the normal gene
The Structure/Function of Ribosomes
ribosoomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis
a ribosome consists of a large subunit and a small subunit, each made up of proteins and one or more
ribosomal RNAs (rRNAs)
in eukaryotes the subunits are made in the nucleolus
ribosomal RNA genes are transcribed, and the RNA is processed and assembled with proteins imported from the cytoplasm
completed ribosomal subunits are then exported by nuclear pores to the cytoplasm
about one-third of the mass of a ribosome is made up of proteins; the rest consists of three rRNA molecules (in bacteria) or four (in eukaryotes)
rRNA is the most abundant type of cellular RNA
eukaryotic ribosomes are slightly larger and differ from bacterial ribosomes in their molecular composition
each ribosome has 3 binding sites for tRNA:
the
P site (peptidyl-tRNA binding site)
holds the tRNA carrying the growing polypeptide chain
the
A site (aminoacyl-tRNA binding site)
holds the tRNA carrying the next amino acid to be added to the chain
the
E site (exit site)
discharged tRNAs leave the ribosome through this
the ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid so that it can be added to the carboxyl end of the growing polypeptide
The Structure/Function of Transfer RNA
each tRNA molecule enables translation of a given of a given mRNA codon into a certain amino acid
it is possible bc tRNA bears a specific amino acid at one end of its 3-D structure, while at the other end is a nucleotide triplet that can base-pair with the complementary codon on mRNA
tRNA is a molecule that has a single strand of RNA that is only about 80 nucleotides long (compared to hundreds of nucleotides for mRNA)
this single strand can fold back on itself and form a molecule with a 3-D structure
the shape for a tRNA molecule looks like a cloverleaf
the tRNA actually twists and folds into a roughly L-shaped 3-D figure with the 5' to 3'ends of the linear tRNA both located near one end of the structure
the protruding 3' end acts as the attachment site for an amino acid
the loop extending from the othe rend of the L includes the
anticodon
, the particular nucleotide triplet that base-pairs to a specific mRNA codon
Translation is the RNA-directed synthesis of a polypeptide
Translation: the basic concept
as a molecule of mRNA is moved through a ribosome, codons are translated into amino acids, one by one. the translators (or interpreters) are tRNA molecules, each with a specific anticodon at one end and a corresponding amino acid at the other end. a tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon hydrogen-bonds ti the complementary codon on the mRNA
a cell "reads" a genetic message and builds a polypeptide accordingly
thus message is a series of codons along an mRNA molecule and the translator is called a
transfer RNA (tRNA)
its function is to transfer an amino acid from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome
the cytoplasm is stocked with all 20 amino acids (either by synthesizing from other compounds or by taking them up from the surrounding solution)
a ribosome adds each amino acid brought to it by tRNA to the growing end of a polypeptide chain
