BBL Lesson 15: Transcription and Translation

Two kinds of interactions that determine where protein synthesis starts in prokaryotes

(1) the pairing of mRNA bases on the Shine-Dalgarno region with the 3′ end of rRNA of the small ribosomal subunit and (2) the pairing of the initiator codon on mRNA with the anticodon of an initiator tRNA molecule known as formylmethionyl-tRNA. Though a Met residue is typically found at the amino-terminal end of proteins expressed in e. coil, it is usually modified to an amino acid known as N-formylmethionine (f Met).

What are the characteristics of the genetic code?

3 nucleotides encode an amino acid, the code is non-overlapping, the code has directionality, the code is nearly universal and the code is degenerate.

Similarities between RNA and DNA Synthesis

1. Direction of RNA synthesis is 5′ → 3′, 2. the mechanism of RNA and DNA elongation is similar in that the 3′-OH group at the terminus of the growing chain attacks the innermost phosphoryl group of the incoming ribonucleoside triphosphate, and 3. the synthesis is driven by the hydrolysis of pyrophosphate.

3 Stages of RNA Synthesis

1. Initiation, 2. Elongation, 3. Termination

tRNA Molecule Characteristics

1. Single strand containing between 73 and 93 ribonucleotides(∼25 kd). 2. Molecule is L-shaped, as shown in the figure above to the right. 3. Contain many unusual bases, typically between 7 and 15 per tRNA, such as methylated or dimethylated derivatives of A, U, C, and G as well as inosine (I). Methylation prevents the formation of certain base pairs and imparts a hydrophobic character on specific regions. Allows the molecules in some cases to interact better with various components of the translational machinery. 4. All molecules can be arranged in a cloverleaf pattern when depicted in a 2D perspective. In 2D representation, approximately half the ribonucleotides in tRNAs are base-paired to form double helices. The five regions not base-paired in this way are as follows: the 3' CCA terminal region which is part of the acceptor stem, the TΨC loop, the "extra arm" which contains a variable number of bases, the the DHU loop, and the anticodon loop. The 5′ end of a tRNA is phosphorylated. 5. Activated amino acid is attached to a hydroxyl group of the adenosine residue located at the end of the CCA component of the acceptor stem, a flexible region at the 3′ end of mature tRNAs. 6. The anticodon loop, which contains the three-base ribonucleotide anticodon, is near the center of the sequence.

Eukaryotic RNA Polymerases

3 different kinds. All these polymerases are large proteins, containing from 8 to 14 subunits, are similar to one another and to prokaryotic RNA polymerase in structure and function. Like prokaryotic genes, eukaryotic genes require promoters for transcription initiation. Depending on the type of RNA polymerase they bind, eukaryotic promoters differ distinctly in sequence and position relative to the regulated genes. The ability of the the various eukaryotic RNA polymerases to recognize specific promoters accounts for the template specificity.

Ribosome Binding Sites

3 tRNA-binding sites on ribosomes that are sequentially arranged to allow the sequential formation of peptide bonds between amino acids attached to the tRNA molecules. The mRNA fragment being translated is bound within the small subunit (30S). Each of the aminoacyl-tRNA molecules is in contact with both the small and large subunits. Two of the three tRNA molecules are bound to the mRNA through anticodon-codon base pairing. These binding sites are referred as the aminoacyl or A site, the peptidyl or P site, and the exit or E site. The other end of each tRNA molecule interacts with the 50S subunit. The acceptor stems of the tRNA molecules occupying the A site and the P site converge at a site where a peptide bond is formed. A tunnel, or polypeptide channel, connects this site to the back of the ribosome through which the polypeptide chain passes during synthesis.

What is a synonym in the genetic code?

A codon that codes for the same amino acid.

Anticodon

A consecutive three base pair region on the tRNA that is complementary to the consecutive three base pair codons of mRNA. This match between the codons of mRNA and anticodons of tRNA allow for the proper amino acids to be inserted into a growing polypeptide chain. The codon and anticodon are aligned in an antiparallel fashion. Recall that nucleotide sequences are written in the 5′ → 3′ direction by convention. Thus, the anticodon to AUG is written as CAU, but the actual base-pairing with the codon would be UAC, as shown below. Some tRNA molecules are able recognize multiple codons. This greater pairing freedom ("wobble") typically is associated with the presence of inosine in the anticodon region which is able to bind to U, C, or A.

Translation: Termination

A release factor binds to the A site at a stop codon and the polypeptide is released from the transfer R.N.A. in the P site. After dissociation the polypeptide may need to be modified before it is ready to function.

RNA Polymerase needs...

A template (antisense DNA strand, active precursors (ATP, GTP, UTP, CTP) and a divalent metal ion (cation cofactor, ex: Mg2+ or Mn2+) to function properly.

What is the start codon?

AUG

Aminoacyl-tRNA

Activated form of tRNA (aka charged tRNA) is formed by linking the amino acid to the tRNA through an ester bond to either the 2′-or the 3′-hydroxyl group of the ribose unit at the 3′ end of tRNA.

Other Regulatory Sequences

Additional regulatory sequences known as the CAAT box and the GC box may be located between positions −40 and −150 base in some genes.

Transcription Factors

Aka trans-acting elements. Regulatory proteins known to have a large impact on the efficiency of transcription by binding to specific sequences near promoter sites and interacting with RNA polymerase.

The physical separation of transcription and translation due to a nucleus allows for what?

Allows for mRNA to undergo extensive post-transcriptional processing and provides eukaryotic organisms a remarkable level of control over which genes are expressed, the time at which each gene is transcribed, and how much RNA is produced.

What does the degeneracy of the code allow for?

Allows for mutations that will not change the encoded amino acid, such as when a mutation produces a synonym. Probability of mutating to chain termination is decreased significantly.

RNA Polymerase Core Enzyme (E. coli)

Alpha, beta, and omega subunits together. This version of the enzyme from e. coli is unable to start transcription at promoter sites. In the absence of the sigma subunit, the core enzyme binds DNA randomly and tightly

tRNA

An adaptor molecule that connects the amino acids to their corresponding codons. Acts by binding to a specific codon and bringing with it an amino acid for incorporation into the nascent polypeptide chain. Each amino acid has at least one to assist it in gaining access to the translational machinery. Share many of the same structural characteristics to be able to interact with the ribosome and mRNA.

Where is the majority of gene expression controlled?

At the promoter region.

Translation: Initiation

Begins with the messenger R.N.A. strand binding to the small ribosomal subunit upstream of the start codon. Each amino acid is brought to the ribosome by a specific transfer R.N.A. molecule. The type of amino acid is determined by the anticodon sequence of the transfer R.N.A. Complementary base pairing occurs between the codon of the messenger R.N.A. and the anticodon of the transfer R.N.A. After the initiator transfer R.N.A. molecule binds to the start codon, the large ribosomal subunit binds to form the translation complex.

Eukaryotic Transcription Factors

Bind to the cis-acting elements. RNA polymerase II is guided to the start site by, TFIIA - TFIIH, known collectively as TFII. Initiation for RNA polymerase II begins when TFIID binds to the TATA box.

Exons

Coding portions of nascent RNA

Spliceosome

Complex of proteins that performs intron splicing. Removes the intransigents and joins the adjacent exon's to produce a mature messenger R.N.A. strand that can leave the nucleus through a nuclear pore and enter the cytoplasm to begin translation.

RNA Polymerase Holoenzyme (E. coli)

Created by the binding of the sigma subunit (cofactor, sigma 70) to the core enzyme to complete the polymerase. This version has a much lower affinity for DNA and can rapidly slide along DNA to search for and recognize the promoter regions and UP element.

What is the main difference between RNA and DNA synthesis?

DNA polymerases require a primer whereas RNA polymerases do not require a primer.

Coding (sense) DNA Strand

Has an identical sequence to that of the RNA transcript (mRNA) except for thymine (T) in place of uracil (U). It itself is not the template though.

Upstream Element (UP)

Exists outside the -10 and -35 promoter regions. This sequence is located between 40 to 60 nucleotides upstream of the transcription start site. It binds to RNA polymerase and thus increases the efficiency of transcription by creating an additional binding site for the polymerase.

RNA Synthesis: Initiation (E. coli)

First step is finding the start site for transcription which depends on the sigma subunit binding and assisting the polymerase locate the correct start site. Once the nascent RNA chain is approximately nine to ten residues in length, the sigma subunit releases and assists another RNA polymerase core enzyme. E. coli has many kinds of sigma factors for recognizing several types of promoter sequences found in the DNA. The sigma factor that that recognizes the consensus sequences is known as sigma 70 because it has a molecular weight of 70 kd.

Which step in RNA synthesis allows for termination to occur?

Formation of a hairpin-oligo(U) structure at the 3' end of the nascent RNA chain.

Enhancer Elements

Iincrease the efficiency of translation by binding transcription factors. These elements do not act on the promoter region, can be very distant (more than 1,000 base pairs) from the start site, and can be located either upstream or downstream from the start site.

Prokaryote vs. Eukaryote RNA Transcription and Translation Locations

In eukaryotes, transcription takes place in the membrane-bound nucleus while translation takes place outside the nucleus in the cytoplasm (or in ER). The spatial and temporal separation of transcription and translation allows eukaryotes to control gene expression in much more intricate ways, contributing to the richness of eukaryotic diversity. In prokaryotes, the two processes are closely coupled. Translation of bacterial mRNA begins while the mRNA transcript is still being synthesized.

Which amino acids are encoded by 6 codons each?

Leucine, arginine and serine.

Eukaryotic Transcription: Initiation

The key initial event is the recognition of the TATA box by the TATA-box-binding protein (TBP), a 30-kd component of the 700-kd TFIID complex. In promoters without TATA boxes, other proteins in the TFIID complex bind the core promoter elements. TBP bound to the TATA box is the core of the TFII initiation complex. TFIIA then joins the complex followed by TFIIB. TFIIF, RNA polymerase II (yellow), TFIIE, and TFIIH bind the complex sequentially to form the the basal transcription apparatus.

Translation: Elongation

In the large ribosomal subunit there are three distinct regions called the E, P, and A sites. Individual amino acids are brought to the messenger R.N.A. strand by a transfer R.N.A. molecule through complementary base pairing of the codons and anticodons. Each anticodon of a transfer R.N.A. molecule corresponds to a particular amino acid. A charged transfer R.N.A. molecule binds to the A site and a peptide bond forms between its amino acid and the one attached to the transfer R.N.A. molecule at the P site. The complex slides down one codon to the right where the now uncharged transfer R.N.A. molecule exits from the E site, and the A side is open to accept the next transfer R.N.A. molecule. Process continues until a stop codon is reached.

Eukaryotic Translation Initiation

Initiating codon in eukaryotes is always AUG, codes for methionine (Met). The 40S small ribosomal unit bound to the Met aminoacyl-tRNA (Met-tRNAi, also sometimes referred to as the initiator tRNA) attaches to the cap at the 5′ end of eukaryotic mRNA and searches for an AUG codon by moving step-by-step in the 3′ direction. This search process is catalyzed by helicases that move along the mRNA and is powered by hydrolysis of ATP. Pairing of the anticodon of Met-tRNAi with the AUG codon of mRNA signals that the target has been found. Eukaryotes also utilize several initiation factors, small proteins that bind to the small ribosomal subunit to assist in the translation process.

Which base is associated with the "wobble" properties of tRNA allowing for greater pairing freedom?

Inosine in the anticodon region, which is able to bind to U, C, or A.

RNA Synthesis: Elongation

R.N.A. polymerase slides along the template D.N.A. strand. As the complementary bases pair up, the R.N.A. polymerase links nucleotides to the 3' end of the growing R.N.A. molecule.

Why is the binding of the sigma subunit to RNA polymerase important to RNA synthesis?

It allows for RNA Polymerase to loosen its grip on DNA enough to find the promoter and enhancer sequences at the start site. It falls off after the RNA chain is around 9 to 10 residues in length.

What is the function of the phosphorylation of CTD by TFIIH in eukaryotic transcription?

It begins the transition from initiation to elongation by opening the DNA double helix and allowing RNA polymerase II to leave the promoter site and begin transcription.

Where is a promoter located in relation to the gene it regulates?

It is located upstream on the same DNA strand of the gene it will regulate (cis-acting element).

Ribosomes

Large molecular assemblies consisting of both proteins and rRNA. In general, can be dissociated into a large subunit and a small subunit. For E. coil, these subunits are referred to as 50S and 30S respectively. In general, eukaryotic ones are larger than prokaryotic ribosomes. However, they still consist of a large subunit (60S) and small subunit (40S) composed of proteins and rRNA molecules and perform the same functions during translation.

RNA Polymerase

Large protein complex that catalyzes the elongation of RNA molecules.

Which two amino acids are only encoded by 1 codon?

Methionine and Tryptophan.

RNA Post-Transcriptional Modification

Methylated cap (5' end, during transcription), splicing out introns by spliceosomes, and addition of PolyA tail (3' end, by PolyA polymerase)

What function does methylation play in the structure of tRNA?

Methylation prevents the formation of certain base pairs and imparts a hydrophobic character on specific regions. Allows the molecules in some cases to interact better with various components of the translational machinery.

TATA Box

Most commonly recognized cis-acting element for genes transcribed by RNA polymerase II, named on the basis of its consensus sequence. It is highly conserved in many eukaryotic organisms. It is typically centered between positions −24 and −32.

Where is the anticodon loop located in a tRNA?

Near the center of the sequence.

Codon

Nitrogenous bases are grouped into 3-letter codes, 64 included in genetic code. 1 start codon (AUG), 3 stop codons (UAA, UAG, UGA). The rest code for specific amino acids.

Introns

Non-coding portions of nascent RNA

RNA Synthesis: Elongation (E. coli)

Occurs in the 5' to 3' direction. The loss of sigma factor enables the core enzyme to strongly bind to the DNA template with RNA polymerase continuing transcription until a termination signal is reached. The cellular region containing RNA polymerase, DNA, and nascent RNA is called a transcription bubble. The transcription bubble catalyzes the elongation of the nascent RNA strand at a rate of approximately 50 nucleotides per second, extruding the newly synthesized RNA from the polymerase.

RNA Synthesis: Termination

Once the R.N.A. polymerase reaches the terminator portion of the gene, the messenger R.N.A. transcript is complete and the R.N.A. polymerase, the D.N.A. strand, and the messenger R.N.A. transcript dissociate from each other.

Intron Splicing

Process where the non-coding introns need to be removed and modifications such as a 5' cap and a 3' poly-A tail are added so that the RNA can be used in translation.

Prokaryote vs. Eukaryote RNA Polymerase and Promoters

RNA synthesis in eukaryotes is carried out by three distinct RNA polymerases. These RNA polymerases rely on promoter sequences in DNA to control the initiation of transcription. As expected, the three different types of polymerases found in eukaryotic organisms greatly increases the number of types of promoters. Adding to this complexity is the fact that not all promoters are recognized by each of the polymerases and thus the eukaryotic promoter elements can combine in a multitude of ways. In contrast, bacteria have just three promoter elements and one type of RNA polymerase.

Eukaryotic RNA Polymerase II

Responsible for transcribing nascent mRNA and several small RNA molecules used in a variety of cellular functions. Promoters include a set of conserved-sequence elements that define the start site by recruiting the polymerase

Eukaryotic RNA Polymerase I

Responsible for transcribing rRNA.

Eukaryotic RNA Polymerase III

Responsible for transcribing tRNA

Shine-Dalgarno Sequence

Sequence in prokaryotic mRNA is centered about 10 nucleotides on the 5′ side of the start codon. It interacts with a complementary sequence on the 3′ end of the rRNA component of the 30S small ribosomal unit to help initiate translation.

RNA Synthesis: Termination (E. coli)

Signaled by the formation of a hairpin-oligo(U) structure at the 3' end of the nascent RNA chain. This structure is formed through the transcription of DNA that is palindromic with a GC-rich region followed by a sequence of T residues. GC-rich region in the RNA transcript forms a stable hairpin followed by the repeat of four or more uracil residues. The folding of the hairpin pauses transcription and the nascent RNA chain is released shortly after as the oligo(U) tail is weakly bound to the template DNA strand. The DNA template strand then rejoins the coding strand to re-form the DNA duplex and the transcription bubble closes.

Promoter

Site of RNA synthesis initiation on DNA template strand. On same molecule of DNA that it regulates (cis-acting element). In bacteria, the two most common DNA sequences that act as promoters for many genes are known as the −10 site (Pribnow box) and the −35 sequence. As shown below, these promoters are centered at approximately 10 and 35 nucleotides respectively upstream of the start site and each are 6 base pairs long. The first nucleotide or the start site of a transcribed DNA sequence is denoted numerically as +1. The strength of a promoter sequence regulates transcription. Genes with strong promoters are transcribed frequently, sometimes as often as every 2 seconds in E. coli, while genes with very weak promoters are transcribed about once in 10 minutes. The −10 and −35 regions of the strongest promoters have sequences that closely resemble the consensus sequences, whereas weak promoters often have several substitutions at these sites. In many instances, the mutation of even a single base in the promoter sites can alter the efficiency of transcription.

Eukaryote Polymerases are more complex than in Prokaryotes in which ways?

There are three distinct RNA polymerases and there are multiple different promoter sequences. However, not every RNA polymerase can interact with every promoter adding an extra level of combination.

Degeneration of Genetic Code

Some amino acids are encoded by more than one codon. There are 64 possible base triplets for the basic 20 amino acids. Tryptophan and methionine are encoded by just one triplet each while the other 18 amino acids are each encoded by two or more. Amino acids such as leucine, arginine, and serine can be coded by six codons each. Codons that specify the same amino acid are called synonyms. 61 of the 64 possible triplets specify particular amino acids and the other three, known as stop codons, designate the termination of translation. AUG, which codes for methionine, also is part of the initiation signal of translation. If the code were not degenerate, only 20 codons would code for amino acids and 44 would lead to chain termination. Allows for mutations that will not change the encoded amino acid, such as when a mutation produces a synonym. Probability of mutating to chain termination is decreased significantly.

Initiator Element (Inr)

TATA box is often paired with this sequence found at the transcriptional start site between positions −3 and +5

Which transcription factors guide eukaryotic RNA polymerase II to the start site?

TFIIA - TFIIH.

How are the A site and P site connected in the ribosome during translation?

The acceptor stems of the tRNA molecules occupying the A site and the P site converge at a site where a peptide bond is formed. A tunnel, or polypeptide channel, connects this site to the back of the ribosome through which the polypeptide chain passes during synthesis.

Aminoacyl-tRNA synthetase

The activating enzyme for an amino acid, typically highly specific for only one amino acid. Next to its active site is its editing site where it quickly excises an incorrect amino acid using the CCA arm of the tRNA to swing back and forth between the sites without disassociating from the enzyme.

What happens in the P site of a Ribosome during translation elongation?

The amino acid (or growing peptide chain) on the tRNA forms a peptide bond in a condensation reaction with the amino acid of the tRNA in the A site and the growing chain is transferred to that tRNA.

Prokaryotic Translation Initiation

The first translated codon is usually more than 25 nucleotides away from the 5′ end. Similar to eukaryotic systems, the the start codon of bacterial mRNA is AUG (methionine). However, in addition to the AUG start codon each initiator region contains a purine-rich sequence known as the Shine-Dalgarno sequence which is centered about 10 nucleotides on the 5′ side of the start codon. Nucleotide sequences in mRNA, such as the Shine-Dalgarno sequence, that are not translated are called untranslated regions (UTRs).

Prokaryote vs. Eukaryote RNA Post-Transcriptional Modification

The post transcriptional processing of RNA occurs in both prokaryotes and eukaryotes; however, eukaryotes extensively modify nascent RNA destined to become mRNA. Modifications to the nascent RNA include changes to both the 5' and 3' ends and splicing out segments of the primary transcript. RNA processing.

Translation

The process by which large, cellular complexes known as ribosomes interpret, or translate, the RNA molecules formed through transcription to produce proteins.

Transcription

The process by which the genetic information stored within DNA is decoded, or transcribed, from a double stranded molecule of DNA into single stranded molecules of RNA.

RNA Synthesis: Initiation

The promoter region of the gene functions as a recognition site for R.N.A. polymerase to bind. This is where the majority of gene expression is controlled, by either permitting or blocking access to this site by the RNA polymerase. Binding causes the D.N.A. double helix to unwind and open

What is the key initial event in eukaryotic transcription?

The recognition of the TATA box by the TATA-box-binding protein (TBP), a 30-kd component of the 700-kd TFIID complex.

Genetic Code

The relationship between the sequence of bases in DNA and their mRNA transcripts to the sequence of amino acids in proteins. Characteristics of the code: 3 nucleotides encode an amino acid, the code is nonoverlapping, the code has directionality, the code is nearly universal, the code is degenerate.

Template (antisense) DNA Strand

The strand that acts as the template for RNA synthesis, usually only one of the strands is transcribed. The sequence of the strand is the complement of that of the transcribed RNA molecule.

What happens in the E site of a Ribosome during translation elongation?

The tRNA now with its amino acid removed and added to the growing peptide leaves the complex.

How are the TFII factors similar to sigma factor in prokaryotic transcription?

They help to initiate transcription but then disassociate as elongation begins (or even before RNA polymerase leaves the promoter as in eukaryotes) and then go on to assist in the initiation of transcription elsewhere.

Eukaryotic Transcription Control

Three types of RNA polymerases. Transcription of RNA occurs in the nucleus of the cell and thus RNA must then be transported across the nuclear membrane into the cytoplasm before translation can occur. This physical separation allows for mRNA to undergo extensive post-transcriptional processing and provides eukaryotic organisms a remarkable level of control over which genes are expressed, the time at which each gene is transcribed, and how much RNA is produced.

What is the elemental classification of transcription factors?

Trans-acting elements.

What are the stop codons?

UAA, UAG, UGA

Eukaryotic Transcription: Transition from Initiation to Elongation

Unlike other RNA polymerases, RNA polymerase II contains a unique carboxyl-terminal domain called the CTD. This domain regulates the activity of RNA polymerase II as it contains multiple serine residues that can be phosphorylated. Phosphorylation of the CTD enhances transcription and recruits other transcription factors required to process the nascent mRNA product. In the formation of the basal transcription apparatus, the CTD is initially unphosphorylated. Phosphorylation of the CTD by TFIIH begins the transition from initiation to elongation by opening the DNA double helix and allowing the polymerase to leave the promoter site and begin transcription. Most of the TFII transcription factors are released before the polymerase leaves the promoter. Similar to sigma factors in prokaryotic transcription, the TFII factors can then participate in sequential rounds of transcription.

Downstream Core Promoter Element (DPE)

When a gene does not contain the TATA box, this is commonly found in conjunction with the Inr. It is found downstream of the start site between positions +28 and +32.

Three Main Types of RNA

messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)