BIOL #13: Protein Synthesis Flashcards
Gene Expression
the process by which DNA directs the synthesis of proteins or RNA molecules
Genotype and Phenotype
All expressed traits (eye color, hair type, etc.) are determined by the synthesis of particular proteins and RNA molecules involved in protein synthesis
- Proteins are the link between genotype (genetic makeup) and phenotype (outward expression of genes)
+ Cells synthesize and breakdown most biological molecules via metabolic pathways. These pathways include a number of enzymes (proteins) and such pathways may result in the synthesis of important molecules (e.g. eye color is a trait that results from the synthesis of pigment in cells of the iris).
Protein Synthesis
The expression of genes that code for proteins involves two main processes:
- Transcription
- Translation
Protein synthesis can result from external signals (pictured here) or internal cellular signals
RNA plays a vital role in protein synthesis:
- RNA contains a ribose sugar rather than a deoxyribose sugar
- RNA contains the bases A, G, C, U (uracil)
- RNA is typically single stranded
Transcription is synthesis of RNA using information in the DNA
Translation is the synthesis of a polypeptide using the information from the transcribed RNA
Prokaryotes vs Eukaryotes
Transcription and translation occurs in all organisms
The basic mechanisms for transcription and translation are similar for bacteria and eukaryotes with one major difference:
- The lack of a nuclear membrane in prokaryotic cells means that transcription and translation is not separated in space and time in bacterial cells – translation of mRNA may begin while it is still being transcribed
Change in Language from DNA to protein
For DNA or RNA (polymers), the monomers are the four types of nucleotides, which differ in nitrogenous bases
For proteins/polypeptides (polymers), the monomers are amino acids
In protein synthesis – DNA monomers must be transcribed then translated into protein monomers
There are 20 different amino acids
How are four DNA bases translated into 20 different amino acids?
The instructions for a polypeptide are written in the DNA as a series of non-overlapping, three-nucleotide words (triplets), called codons.
Transcription
The first step in converting genetic information into proteins is transcription – the synthesis of an RNA version of the instructions stored in DNA.
The DNA strand to be transcribed is called the template strand
The RNA strand built to be complementary to the template strand is called messenger RNA (mRNA)
- Note: mRNA uses uracil (U) in place of thymine (T)
Transcription involves three stages:
1) Initiation
2) Elongation
3) Termination
Initiation (Transcription)
Initiation is the first stage of transcription:
- RNA polymerase begins transcription by prying the two strands of DNA apart and synthesizing the mRNA strand based on the template strand
While RNA polymerases do not require primers to begin transcription, they cannot initiate transcription on their own.
RNA polymerases must attach to promoters to begin transcription
- Promoters are specific DNA sequences that come before the start point of transcription (i.e. upstream of the transcription site)
- RNA polymerase binds in a precise location and orientation on the promoter, which determines where transcription starts and which of the two strands of the DNA helix is used as the template
RNA Polymerase
Like the DNA polymerases, an RNA polymerase can only perform template-directed synthesis in the 5′ to 3′ direction.
Unlike DNA polymerases, RNA polymerases do not require a primer to begin synthesis.
Bacteria have one RNA polymerase while eukaryotes have three distinct types, RNA polymerase I, II, and III.
Initiation & Promoters: Bacteria vs Eukaryotes
In bacteria, the RNA polymerase itself can recognize and bind to the promoter region
In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription
- Many of the eukaryotic promoters include a unique sequence called the TATA box, centered about 30 base pairs upstream of the transcription start site.
- Only after transcription factors are attached to the promoter can RNA polymerase II bind to the promoter
- The transcription factors plus RNA polymerase bound to the promoter is called the transcription initiation complex
Elongation (Transcription)
During the elongation phase of transcription, RNA polymerase moves along the DNA template and synthesizes RNA in the 5’ 3’ direction.
Termination (Transcription)
The mechanisms of termination differ between bacteria and eukaryotes:
In bacteria, there is a terminator sequence in the DNA that causes the RNA polymerase to detach from the DNA and release the mRNA transcript.
In eukaryotes, RNA polymerase transcribes a sequence on the DNA called the polyadenylation signal sequence, which codes for a polyadenylation signal (AAUAAA) in the mRNA transcript. At a point ~10-35 nucleotides downstream from the AAUAAA signal, proteins associated with the growing mRNA transcript cut and release the transcript.
RNA Processing
In bacteria, the information in DNA is converted directly to mRNA.
In eukaryotes, the product of transcription is an immature primary transcript, or pre-mRNA. Before primary transcripts can be translated, they have to be processed in a complex series of steps.
- Specific enzymes are involved in the RNA processing that produces a mature mRNA:
+ Both ends of the pre-mRNA are altered
+ Certain interior sections of the pre-mRNA are cut out and the remaining sections are spliced together
Exons vs. Introns
The protein-coding regions of eukaryotic genes are interrupted by noncoding regions.
- To make a functional mRNA, these noncoding regions must be removed.
Exons are the coding regions of eukaryotic genes that will be part of the final mRNA product.
The intervening noncoding sequences are called introns, and are not in the final mRNA.
RNA Splicing
The transcription of eukaryotic genes by RNA polymerase generates a primary RNA transcript (pre-mRNA) that contains exons and introns.
- Introns are removed by splicing.
Short nucleotide sequences at each end of an intron signal splicing sites
Small nuclear ribonucleoproteins (snRNPs) recognize these splice sites.
snRNPs plus proteins form a complex called a spliceosome, which catalyzes the splicing reaction.
Alternative RNA Splicing
Some introns might contain sequences that regulate gene expression or affect gene products.
- The prevalence of such functions are still under debate
An important consequence of the presence of introns in genes is that a single gene can encode more than one kind of polypeptide:
- Many genes can give rise to two or more different polypeptides, depending on which segments are treated as exons during RNA processing – this is called alternative RNA splicing
+ Example: sex differences in fruit flies are mostly due to differences in how males and females splice the RNA transcribed from particular genes
Caps and Tails
RNA processing involves the addition of a 5′ cap and a poly(A) tail.
The 5’ cap serves as a recognition signal for the translation machinery.
- Consists of a modified guanine nucleotide
The poly(A) tail extends the life of an mRNA by protecting it from degradation.
The UTRs (untranslated regions) of the mature mRNA will not be translated into protein but have other functions, such as RNA binding
Translation
In translation, the sequence of bases in the mRNA is converted to an amino acid sequence in a protein.
A cell keeps its cytoplasm stocked with all 20 amino acids
Ribosomes are structures made of RNA and protein, which catalyze translation of the mRNA sequence into protein.
Transfer RNAs (tRNAs) act as translators, transferring amino acids from the cytoplasmic pool to a growing polypeptide in the ribosome.
transfer RNA (tRNA)
Francis Crick first proposed that an adapter molecule holds amino acids in place while interacting directly and specifically with a codon in mRNA
The adapter molecule was later found to be a small RNA called transfer RNA (tRNA)
The characteristic 3-dimensional shape of tRNAs is an upside-down L-shape
Each tRNA binds to its amino acid at one end (amino acid attachment site)
The other end of the tRNA consists of an anticodon, which base-pairs with a complementary codon on mRNA
Each tRNA translates a particular RNA codon into a particular amino acid.
- Each tRNA anticodon complements a particular mRNA codon.
tRNA Origin
tRNAs are transcribed from DNA templates, like mRNA, thus they are made in the nucleus.
They travel out of the nucleus to the cytoplasm where translation occurs.
Each tRNA molecule is used repeatedly, picking up its designated amino acid in the cytosol and depositing it along the growing polypeptide chain at the ribosome.
aminoacyl-tRNA synthetases
The correct matching up of a tRNA and amino acid is carried out by a family of enzymes called aminoacyl-tRNA synthetases
- The active site of each aminoacyl tRNA synthetase fits only a specific combination of amino acid and tRNA
There are 20 different synthetases, one for each amino acid
- Each synthetase is a able to bind to all the different tRNAs that code for its particular amino acid
Linkage of the tRNA and amino acid with a covalent bond is an endergonic reaction that requires ATP
When the amino acid is bonded to the tRNA it is called an aminoacyl tRNA or charged tRNA
Wobble Hypothesis
There are 61 different codons but only about 40 tRNAs in most cells.
To resolve this deficit, Francis Crick proposed the wobble hypothesis. This hypothesis states that the anticodon of tRNAs can still bind successfully to a codon whose third position requires a nonstandard base pairing.
Thus, one tRNA is able to base pair with more than one type of codon.
rRNA
rRNAs (ribosomal RNAs) group with proteins to form the subunits of ribosomes
Ribsomes are the structure that facilitate coupling of tRNA anticodons with mRNA codons during protein synthesis
- Catalyze the formation of peptide bonds between the amino acids
Genes for rRNA are transcribed and then the subunits and proteins are assembled in the nucleolus. The structures are then exported to the cytoplasm.
Binding Sites
The large and small subunits of ribosomes only come together when they attach to a mRNA molecule
Each ribosome has three binding sites for tRNAs:
- A site: holds the tRNA carrying the next amino acid to be added to the polypeptide chain
- P site: holds the tRNA carrying the growing polypeptide chain
- E site: discharges tRNA from the ribosome
Initiation (Translation)
First, a small ribosomal subunit binds to both mRNA and a specific initiation tRNA
- The codon AUG is the start codon that initiates translation, the specific initiation tRNA carries the corresponding amino acid – methionine
This attachment is followed by the attachment of the large ribosomal subunit, resulting in formation of a translation initiation complex,
- The cell expends energy obtained by GTP hydrolysis to form this complex
Proteins called initiation factors are required to bring all of these components together
At the end of initiation the initiator tRNA sits in the p site
Elongation (Translation)
During elongation, amino acids are added one by one to the previous amino acid of the growing chain
- The mRNA is moved through the ribosome in the 5’ to 3’ direction
Each addition requires the participation of several proteins called elongation factors
Elongation of the polypeptide chain occurs in a three-step cycle
1) Codon Recognition
2) Peptide Bond Formation
3) Translocation
Steps 1 and 3 require energy expenditure (GTP hydrolysis)
Codon Recognition
Codon Recognition:
- The anticodon of an incoming ‘charged’ tRNA basepairs with the complementary mRNA codon in the A site
- Although not shown in the diagram, many different ‘charged’ tRNAs are present but only the one with the appropriate anticodon will bind and allow the cycle to progress
Codon recognition requires hydrolysis of one GTP molecule to increase the efficiency and accuracy of this step
Peptide Bond Formation
An RNA molecule in the large ribosomal subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the growing polypeptide in the P site
The polypeptide is removed from the tRNA in the P site and attached to the tRNA in the A site
Translocation
The ribosome moves the tRNA in the A site to the P site
At the same time the empty tRNA in the P site is moved to the E site and release
This step requires energy from hydrolysis of a GTP molecule
Termination (Translation)
Elongation continues until a ‘stop’ codon in the mRNA reaches the A site of the ribosome
- The codons UAG, UAA, UGA do not code of amino acids, but rather signal to stop translation
- A release factor, a protein shaped like a ‘charged’ tRNA binds directly to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid – this hydrolyzes the chain, releasing the polypeptide through the exit tunnel of the large ribosomal subunit
Polyribosomes
A single ribosome can make an average-sized polypeptide in less than a minute
In most cases, multiple ribosomes translate an mRNA at the same time
- A single mRNA is used to make many copies of a polypeptide simultaneously – once one ribosome is far enough past the start codon, a second ribosome can attach to the mRNA strand –this can result in a string of ribosomes translating a single mRNA strand (polyribosomes)
- This mechanism allows for the creation of a large amount of a single protein
