BIO 161 ch. 17 - Transcription, RNA processing, and Translation

Translation in Bacteria and Eukaryotes

*Table 17.1 af In bacteria, ribosomes attach to mRNA transcripts and begin translation while RNA polymerase is still transcribing the DNA template strand. (Transcription and translation can occur concurrently becasue there is no nuclear envelope to separate the two proceses) Multiple ribosomes attach to each mRNA, forming a polyribosome (many copies of a protein can be produced from a single mRNA) In eukaryotes, primary transcripts are processed in the nucleus to produce a mature mRNA, which is then exported to the cytoplasm. Tx and tl are separate in time and space. Once mRNAs are outside the nucleus, ribosomes can attach to them and behind translation, forming polyribosomes.

How are amino acids attached to tRNA?

-An input of energy (ATP) is required to attach an amino acid to a tRNA -Enzymes called aminoacyl-tRNA snythetases catalyzes the addition of amino acids to tRNAs - what biologists call "charging" a tRNA -For each of the 20 major amino acids, there is a different aminoacyl-tRNA synthetase and one or more tRNAs. Each aminoacyl-tRNA snythetase has a binding site for a particular amino acid and a particular tRNA. Subtle differences in tRNA shape and base sequence allow the enzymes to recognize the correct tRNA for the correct amino acid. The combination of a tRNA molecule covalently linked to an amino acid is called an aminoacyl tRNA.

Transcription in Eukaryotes

-Eukaryotes have three polymerases - RNA polymerase I, II, and III (pol I, pol II, and pol III). Each polymerase transcribes only certain types of RNA in eukaryotes. RNA pol II is the only polymerase that transcribes protein-coding genes. -Promoters in eukaryotic DNA are more diverse than bacterial promoters. Most euk. promoters include a sequence called the TATA box, centered about 30 base pairs upstream of the transcription start site, and other important sequences that vary more widely -Instead of using a sigma protein, euk. RNA polymerases recognize promoters using a group of proteins called basal transcription factors (BTFs). BTFs assemble at the promoter, and RNA polymerase follows -Termination of euk. protein-coding genes involves a short sequence called the polyadenylation signal or poly(A) signal. Soon after the signal is transcribed, the RNA is cut by an enzyme downstream of the poly(A) signal as the polymerase continues to transcribe the DNA template. Eventually, RNA polymerase falls off the DNA template and terminates transcription. -Bacteria end transcription at a distinct site for each gene, but in euks, transcription ends variable distances from the poly(A) signal

Brief summary/overview

-Transcription initiation depends on interactions between proteins associated with RNA polymerase and a promoter sequence in DNA -In bacteria, sigma protein binds to RNA polymerase and contacts the promoter. In eukaryotes, basal transcription factors bind to the promoter and recruit RNA polymerase -During transcription elongation, ribonucleoside triphosphate are the substrate for a polymerization reaction catalyzed by RNA polymerase. The enzyme adds ribonucleotides that are complementary to the template DNA strand. -Transcription ends in bacteria when a termination signal at the end of the gene is transcribed, leading to the dissociation of RNA polymerase and the DNA template

Overview/Summary of Translation with Ribosomes

-Translation begins when (1) the ribosome binding site on an mRNA binds to an rRNA sequence in the small ribosomal subunit, (2) the initiator aminoacyl tRNA binds to the start codon in the mRNA, and (3) the large subunit of the ribosome attaches to the small subunit to complete the ribosome. -Translation elongation occurs when (1) an appropriate aminoacyl tRNA enters the A site, (2) a peptide bond forms between the amino acid held by that tRNA in the A site and the polypeptide held by the tRNA in the P site, and (3) the ribosome moves the mRNA one codon. -Translation ends when the ribosome reaches a stop codon. -Completed proteins are modified by folding and, in many cases, addition of sugar, lipid, or phosphate groups.

Process: Elongation of Polypeptides during Translation (Fig. 17.16)

1. Incoming aminoacyl tRNA: New tRNA moves into A site, where its anticodon base pairs with the mRNA codon. 2. Peptide bond formation: The amino acid attached to the tRNA in the P site is transferred to the amino acid of the tRNA in the A site. 3. Translocation: The ribosome moves one codon down the mRNA with the help of elongation factors. The tRNA attached to the polypeptide chain moves to the P site. The A site is empty. 4. Incoming aminoacyl tRNA: New tRNA moves into A site, where its anticodon base pairs with the mRNA codon. 5. Peptide-bond formation: The polypeptide chain attahed to the tRNA in the P site is transferred to the aminoacyl tRNA in the A site. 6. Translocation: The ribosome once again moves one codon down the mRNA. The tRNA attached to polypeptide chain moves into P site. Empty tRNA from P site moves to E site, where tRNA is ejected. The A site is empty again.

Process: Terminating Translation (Overview)

1. Release factor binds to stop codon, and the release factor fills the A site, breaking the bond linking the tRNA in the P site to the polypeptide chain. 2. Polypeptide and uncharged tRNAs are released. 3. Ribosome subunits separate, and they are ready to attach to the start codon of another message.

The Four Steps of Splicing

1. The process begins when snRNPs bind to the 5' exon-intron boundary, which is marked by the bases GU, and to a key adenine ribonucleotide (A) near the end of the intron. (snRNPs bind to start of intron and an A base within the intron) 2. Once the initial snRNPs are in place, other snRNPs arrive to form a multipart complex called a spliceosome. The spliceosomes found in human cells contain about 145 different proteins and RNAs, making them the most complex macromolecular machines known. (snRNPs assemble to form the sliceosome) 3. The intron forms a loop plus a single-stranded stem (lariat) with the adenine at its connecting point. (Intron is cut; loop forms) 4. The lariat is cut out, and a phosphodiester linkage links the exons on either side, producing a continuous coding sequence - the mRNA. (Intron is released as a lariat; exons are joined together)

Initiating Translation

1. mRNA binds to small subunit. The ribosome binding site sequence binds to a complementary sequence in an RNA molecule in the small subunit of the ribosome, helped by initiation factors (proteins) 2. Initiator aminoacyl tRNA binds to start codon. 3. Large subunit of ribosome binds, completing ribosome assembly. Translation can now begin.

The Structure and Function of tRNA

Amino acids are transferred from tRNAs to proteins (growing polypeptide) - inverse relationship A CCA sequence at the 3' end of each tRNA molecule offered a site for amino acid attachment, while a triplet on the loop at the other end could serve as an anticodon - a set of three ribonucleotides that forms base pairs with the mRNA codon. (The anticodon is at one end, while the CCA sequence and attached amino acid are at the other end) All of the tRNAs in a cell have the same general structure, shaped like an upside-down L. They vary at the anticodon attached amino acid

Adding Caps and Tails to Transcripts

As soon as the 5' end of a eukaryotic pre-mRNA emerges from RNA polymerase, enzymes add a structure called the 5' cap. The cap consists of a modified guanine nucleotide with three phosphate groups. An enzyme cleaves the 3' end of the pre-mRNA downstream of the poly(A) singal. Another enzyme adds a long row of 100-250 adenine nucs that are not encoded on the DNA template strand. This string of adenines is known as the poly(A) tail. With the addition of the cap and tail and completion of splicing, processing of the pre-mRNA is complete. The product is a mature mRNA. Not long after the caps and tails on eukaryotic mRNAs were discovered, evidence began to accumulate that they protect mRNAs from degradation by ribonucleases-enzymes that degrade RNA-and enhance the efficiency of translation. RNA processing is the general term for any of the modifications, such as splicing or poly(A) tail addition, needed to convert a primary transcript into a mature RNA.

Elongation: Extending the PolyPeptide (Intro)

At the start of elongation, the E and A sites in the ribosome are empty of tRNAs. As a result, an mRNA codon is exposed in the A site. Elongation proceeds when an aminoacyl tRNA binds to the codon in the A site by complementary base pairing between the anticodon and the codon. When both the P site and the A site are occupied by tRNAs, the amino acids on the tRNAs are in the ribosome's active site. This is where the peptide-bond formation-the essence of protein synthesis-occurs.

Bacterial Promoters

DNA that is located in the direction RNA polymerase moves during transcription is said to be downstream from the point of reference; DNA located in the opposite direction is said to be upstream. -10 box and -35 box (Fig 17.2b)

Is the Ribosome an Enzyme or a Ribozyme?

Debate over whether ribosomes active site consists of protein or RNA - later discovered that the active sites consists entirely of ribosomal RNA. Protein synthesis is catalyzed by RNA. The ribosome is a ribozyme - not a protein-based enzyme.

Specific structure of ribosomes

During protein synthesis, three distinct tRNAs are lined up inside the ribosome. All three are bound to their corresponding mRNA codons. (Fig. 17.14) The tRNA carrying an amino acid is called the "A" site - A for acceptor or aminoacyl. (rightmost) The tRNA that holds the growing chain occupies the "P" site, for peptidyl, inside the ribosome (P for peptide-bond formation) (Middle) The tRNA that no longer has an amino acid attached and is about to leave, occupies the ribosomes "E" site for exit. (Leftmost)

An Overview of Transcription

Enzymes called RNA polymerase are responsible for synthesizing mRNA. Only one of the two DNA strands is used as a template and transcribed, or "read", by RNA polymerase. -The strand that is read by the enzyme is the template strand. -The other strand is called the non-template strand or coding strand. Coding strand is an appropriate name because, with one exception, its sequence matched the sequence of the RNA that is transcribed from the template strand and codes for a polypeptide. Like DNA polymerase, an RNA polymerase performs a template-directed synthesis in the 5' to 3' direction. But unlike DNA polymerases, RNA polymerases do not require primer to begin transcription.

Overview of what just happened

Eukaryotic genes consist of exons, which are parts of the primary structure that remain in mature RNA, and introns, which are regions of the primary transcript that are removed in forming mature RNA Macromolecular machines, called spliceosomes, splice introns out of pre-mRNAs. Enzymes add a 5' cap and a poly(A) tail to spliced transcripts, producing a mature mRNA that is ready to be translated

Post-Translational Modifications

Even after termination, proteins are not done - most proteins go through an extensive series of processing steps, called post-translational modifications, before they are completely functional. Folding: -Proteins function dependent on shape, and shape depends on how the protein folds. Although folding an occur spontaneously, it is frequently speeded up by proteins called molecular chaperones. Chemical modifications: In the rough ER and Golgi apparatus, small chemical groups may be added to proteins - often sugar or lipid groups that are critical for normal functioning. In addition, many completed proteins are altered by enzymes that add or remove a phosphate group (phosphorylation and dephosphorylation)

RNA Splicing

Fig 17.6 The transcription of eukaryotic genes by RNA polymerase generates a primary transcript that contains both exons and introns. As transcription proceeds, the introns are removed from the growing RNA strand by a process known as splicing. In this phase, pieces of the primary transcript are removed and the remaining segments are joined together. Splicing occurs within the nucleus while transcription is still under way and results in an RNA that contains an uninterrupted genetic message. Splicing of primary transcripts is catalyzed by RNAs called small nuclear RNAs (snRNAs) working with a complex of proteins. These protein-plus-RNA macromolecular machines are known as small nuclear ribonucleoproteins, or snRNPS (snurps).

How does an mRNA triplet specify an amino acid?

Hypothesis 1: Amino acids interact directly with mRNA codons. Hypothesis 2: Adapter molecules hold amino acids and interact with mRNA codons. (This is the correct hypo) Fig 17.10

Events Inside the Haloenzyme (Still Talking About Bacteria)

In bacteria, transcription begins when sigma, as part of the haloenzyme complex, binds to the -35 and -10 boxes. Sigma, and not RNA polymerase, makes the initial contact with DNA of the promoter. Sigma's binding to a promoter determines where and in which direction RNA polymerase will start synthesizing DNA. RNA polymerase is exergonic and spontaneous because NTPs have significant potential energy, owing to their three phosphate groups Figure 17.3

RNA Processing in Eukaryotes

In bacteria, when transcription terminates, the result is a mature mRNA thats ready to be translated into a protein. In fact, translation often begins while the mRNA is still being transcribed. When eukaryotic genes of any type are transcribed, the initial product is termed a primary transcript. This RNA must undergo multistep processing before it is functional. For protein-coding genes, the primary transcript is called pre-mRNA

Initiation: How Does Transcription Begin in Bacteria?

In the initiation phase of transcription, the RNA polymerase must know where and in which direction to start transcription. The enzyme RNA polymerase cannot initiate transcription on its own. Instead, a detachable protein subunit called sigma must bind to the polymerase before transcription can begin. Bacterial RNA polymerase and sigma form a holoenzyme. A holoenzyme consists of a core enzyme (RNA polymerase, in this case), which contains the active site for catalysis, and other required proteins (such as sigma). Sigma could bind to any sequence of DNA. When sigma is added, the holoenzyme formed and bound only to specific sections of DNA. These binding sites were named promoters, because they are sections of DNA that promote the start of transcription.

Elongation and Termination (Bacteria still)

One RNA polymerase begins moving along the DNA template synthesizing RNA, the elongation phase of transcription is under way. During this elongation phase, all the prominent channels and grooves in the enzyme are filled. Double stranded DNA goes into and out of one groove, ribonucleoside triphosphates enter another, and the growing RNA strand exits to the rear - the enzyme's structure is critical for this function Termination ends transcription. In bacteria, transcription stops when RNA polymerase transcribes a DNA sequence that functions as a transcription-termination signal Figure 17.4 - The bases that make up the termination signal are trascribed into a stretch of RNA that folds back on itself and forms a sort double helix that is held together by complementary base pairing (hairpin) This hairpin structure disrupts the interaction between RNA polymerase and the RNA transcript, resulting in the physical separation of the enzyme and its product

Intro to Exons and Introns

Regions of the eukaryotic genes that are part of the final mRNA are exons, while the sections of primary transcript not in mRNA are introns. Introns are sections of genes that are not represented in the final RNA product, ultimately making eukaryotic genesmuch larger than their corresponding mature RNAs (Fig 17.5)

The Structure and Function of Ribosomes

Review: protein synthesis occurs when the sequence of bases in an mRNA is translated into a sequence of amino acids in a polypeptide. The translation of each mRNA codon begins when the anticodon of an aminoacyl tRNA binds to the codon. Translation of a codon is complete when a peptide bond forms between the tRNA's amino acid and the growing polypeptide chain These events occur inside a ribosome - ribosomes contain many proteins and ribosomal RNAs (rRNAs). Ribosomes can be separated into two major structures, the large subunit and small subunit. The small subunit holds the mRNA in place during translation; the large subunit is where the peptide-bond formation takes place.

Ribosomes: The site of protein synthesis

Strong correlation between the number of ribosomes in a given type of cell and the rate at which that cell synthesizes proteins - indicating that ribosomes are the site of protein synthesis Pulse-chase experiment: labels a population of molecules as they are being produced, so that the location of the tagged molecules is then followed over time In this case, the ragging was done by supplying a pulse of radioactive sulfur atoms that would be incorporated into the amino acids methionine and cysteine followed by a chase of unlabeled sulfur atoms... etc.

Terminating Translation

The genetic code include three stop codons. In most cells, no aminoacyl tRNA has an anticodon that bonds to those sequences. When the translocating ribosome reaches one of the stop codons, a protein called a release factor recognizes the stop codon and fills the A site. Release factors fit tightly into the A site since they have the size/shape of a tRNA, however, they do not carry amino acid. When a release factor occupies the A site, the protein's active site catalyzes the hydrolysis of the bond that links the tRNA in the P site to the polypeptide chain, a reaction that frees the polypeptide. The newly synthesized polypeptide and uncharged tRNAs are released from the ribosome, the ribosome separates from the mRNA, the two ribosomal subunits dissociate, for they are ready to attach to the stat codon of another message and start translation anew. Termination occurs in similarly in bacteria and eukaryotes.

Three step sequence of synthesizing proteins

The ribosome is a macromolecular machine that synthesizes proteins in a three-step sequence: 1. An aminoacyl tRNA diffuses into the A site; if its anticodon matches a codon in mRNA, it stays in the ribosome. 2, A peptide bond forms between the amino acid held by the aminoacyl tRNA in the A site and the growing polypeptide, which was held by a tRNA in the P site. 3. The ribosome moves down the mRNA by one codon, and all three tRNAs move one position within the ribosome. The tRNA in the E site exits; the tRNA in the P site moves to the E site; and the tRNA in the A site switches to the P site. -The protein that is being synthesized grows by one amino acid each time this three-step sequence repeats. -Protein synthesis starts at the amino acid end (N-terminus) of a polypeptide and proceeds to the carboxy end (C-terminus).

Translation Intro

To synthesize a protein, the sequence of bases in mRNA is translated into a sequence of amino acids in a polypeptide. The genetic code specifies the correspondence between each triplet codon in mRNA and the amino acid it codes for.

Moving down the mRNA

When the peptide-bond formation is complete, the polypeptide chain is transferred from the tRNA in the P site to the amino acid held by the tRNA in the A site. Translocation occurs when proteins called elongation factors help move the ribosome relative to the mRNA so that translation occurs in the 5' to 3' directions. Translocation is an energy demanding event that requires GTP. Translocation does several things: It moves the uncharged RNA into the E site; it moves the tRNA containing the growing polypeptide into the P site; and it opens the A site and exposes a new mRNA codon. The empty tRNA that finds itself in the E site is ejected into the cytosol. The three steps of elongation: 1. arrival of aminoacyl tRNA 2. peptide-bond formation 3. translocation Repeat down the length of the mRNA