Biology Chapter 17

Which three statements correctly describe the processing that takes place before a mature mRNA exits the nucleus?

A poly-A tail (50-250 adenine nucleotides) is added to the 3' end of the pre-mRNA. Noncoding sequences called introns are spliced out by molecular complexes called spliceosomes. A cap consisting of a modified guanine nucleotide is added to the 5' end of the pre-mRNA.

Anticodon

A set of three ribonucleotides that forms base pairs with the mRNA codon.

Bacteria

A single RNA polymerase Alternative sigma proteins that bind to promoters with slightly different DNA base sequences and may activate a group of genes in response to environmental change Transcription begins when sigma - as part of the holoenzyme complex - binds to the -35 and -10 boxes. Sigma, not RNA polymerase, makes initial contact with DNA of the promoter Sigma binding to a promoter determines where and in which direction RNA polymerase will start synthesizing RNA

RNA processing

Any of the modifications needed to convert a primary transcript into a mature RNA

Eukaryotes

At least three types of RNA polymerase

Initiation Factors

Help in preparing the ribosome for translation, including binding the first aminoacyl tRNA to the ribosome Also prevent the small and large subunits of the ribosome from coming together until the initiator tRNA is in place at the AUG start codon, and they help bind the mRNA to the small ribosomal subunit.

Why does cordycepin end transcription?

It lacks a 3' OH.

Exons

Part of the final mRNA, because they at EXpressed

Initiation

Sigma must bind to the polymerase before transcription can begin, bacterial RNA polymerase and sigma form a holoenzyme

-10 box

Similar to TATAAT, centered about 10 bases from the point where bacterial RNA polymerase starts transcription Centered about 10 bases upstream from the transcription start site

Molecular Chaperones

Speed up folding of proteins

Template Strand

Strand that is read by the enzyme

-35 box

TTGACA 35 bases upstream from the +1 site

Aminoacyl tRNA

The combination of a tRNA molecule covalently linked to an amino acid

What is recognized by an aminoacyl tRNA synthetase?

one amino acid and the set of tRNAs that are coupled to that amino acid

Wobble Hypothesis

1. Many amino acids are specified by more than one codon 2. Codons for the same amino acid tend to have the same nucleotides at the first and second positions but a different nucleotide at the third position Limited flexibility in base pairing Allows just 40 or so tRNA to bind to all 61 mRNA codons

Translation in bacteria steps

1. The mRNA binds to a small ribosomal subunit 2. The initiator aminoacyl tRNA bearing f-met binds to the start codon 3. The large ribosomal subunit binds, completing the complex

Which components of the eukaryotic transcript shown below will also be found in mRNA in the cytosol? Untranslated region (UTR) Exon (E) Intron (I) 5' UTR E1 I1 E2 I2 E3 I3 E4 UTR 3'

5' UTR E1 E2 E3 E4 UTR 3'

Ribozyme

Active sites consist entirely of ribosomal RNA Convinced that protein synthesis is catalyzed by RNA The ribosome is a ribozyme - not a protein-based enzyme

How are amino acids attached to tRNAs?

An input of energy, in the form of ATP, is required to attach an amino acid to a tRNA Enzymes called aminoacyl tRNA synthetases catalyze 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

Caps and Tails

As soon as the 5' end of a eukaryotic pre-mRNA emerges from RNA polymerase, enzymes add a structure called the 5' cap - consisting of a modified guanine nucleotide with three phosphate groups An enzyme cleaves the 3' end of the pre-mRNA downstream of the poly (A) signal. Another enzyme adds a long row of 100-250 adenine nucleotides 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 Caps and tails protect mRNAs from degradation by ribonucleases - enzyme that degrade RNA - and enhance the efficiency of translation

RNA splicing

As transcription proceeds, the introns are removed from the growing RNA strand by a process known as splicing. Pieces of the primary transcript are removed and the remaining segments are joined together. Splicing occurs within the nucleus while transcription is still underway and results in an RNA that contains an uninterrupted genetic message. 1. snRNPs bind to start of intron and an A base within the intron (5' exon-intron boundary, which is marked by the bases GU, and to a key adenine ribonucleotide (A) near the end of the intron) 2. snRNPs assemble to form the spliceosome (other snRNPs arrive to form a multipart comple called spliceosome. The spliceosomes found in human cells contain about 145 different proteins and RNAs) 3. Intron is cut; loop forms (a loop plus a single-stranded stem - a lariat - with adenine at its connecting point) 4. Intron is released as a lariat; exons are joined together (the lariat is cut out and a phosphodiester linkage links the exons on either side, producing a continuous coding sequence - the mRNA) Splicing of primary transcripts is catalyze 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 Reactions are catalyzed by a ribozyme

The proteome is all the proteins produced by an organism. The genome is the totality of all genes of an organism. If the proteome is much larger than the genome, which of the following statements would be accurate?

At least in some cases, a single gene must code for more than one protein.

What determines which base is to be added to an RNA strand during transcription?

Base pairing between the DNA template strand and the RNA nucleotides

Which of the following terms best describes the relationship between the newly synthesized RNA molecule and the DNA template strand?

Because the template strand determines the nucleotides to be added to the RNA strand, using the same complementarity rules of the DNA, they will be complementary to each other.

Downstream

DNA located in the direction RNA polymerase moves during transcription

Upstream

DNA located in the opposite direction that RNA polymerase moves during transcription

Translation

During translation, new amino acids are added one at a time to the growing polypeptide chain. The addition of each new amino acid involves three steps: Binding of the charged tRNA to the A site. This step requires correct base-pairing between the codon on the mRNA and the anticodon on the tRNA. Formation of the new peptide bond. In the process, the polypeptide chain is transferred from the tRNA in the P site to the amino acid on the tRNA in the A site. Movement of the mRNA through the ribosome. In this step, the discharged tRNA shifts to the E site (where it is released) and the tRNA carrying the growing polypeptide shifts to the P site.

Aminoacyl-tRNA synthetase

Each aminoacyl-tRNA synthetase 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

Termination

End of transcription, in bacteria - transcription stops when RNA polymerase transcribes a DNA sequence that functions as a transcription-termination signal As soon as the signal is synthesized, this portion of the RNA folds back on itself and forms a short double helix that is held together by complementary base pairing, this is called a hairpin. This results in the physical separation of the enzyme and its project.

Polymerases

Eukaryotes have three - RNA polymerase I, II, and III (pol I, pol II, 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 eukaryotic promoters include the TATA box sequence, centered about 30 base pairs upstream of the transcription site. Eukaryotic polymerases recognize promoters using a group of proteins called basal transcription factors. Basal transcription factors assemble at the promoter, and RNA polymerase follows. Termination - poly (A) signal, after the signal is transcribed the RNA is cut by an enzyme downstream.

NTP

Has a hydroxyl (OH-) group on the 2' carbon (ribose instead of deoxyribose)

Phases of Transcription

Initiation, elongation, termination

What happens to RNA polymerase II after it has completed transcription of a gene?

It is free to bind to another promoter and begin transcription.

Non-Template/Coding Strand

Its sequence matches the sequence of the RNA that is transcribed from the template strand and codes for a polypeptide (U instead of T in RNA vs. DNA)

Discovery of Introns

Loops in the micrograph representing regions of DNA that are transcribed but are not found in the final mRNA

Polymerization Reaction

NTP matches a base on the DNA template is in place, RNA polymerase cleaves off two phosphates and catalyzes the formation of a phosphodiester linkage between the 3' end of the growing mRNA chain and the new ribonucleoside (tide withough a phosphate group) monophosphate. As this 5'-->3' matching-and-catalysis process continues, an RNA that is complementary to the gene to the gene is synthesized. This is transcription.

Transcription

Nucleotides are added to the 3' end of the growing RNA molecule, During elongation, the RNA strand is extended in the 5' to 3' direction.

Translocation

Occurs when proteins called elongation factors help move the ribosome relative to the mRNA so that translocation occurs in the 5'-->3' direction. An energy-demanding event that requires GTP. Moves uncharged RNA into the E site Moves the tRNA containing the growing polypeptide into the P site Opens the A site and exposes a new mRNA codon The empty tRNA that finds itself in the E site is ejected to the cytosol

What would occur if a mutation caused an aminoacyl-tRNA synthetase to recognize both its normal amino acid and a different one?

One or more codons would sometimes be misread. The tRNA(s) recognized by this aminocacyl-tRNA synthetase could be attached to either amino acid, so the corresponding codon(s) could be read as either the correct or incorrect amino acid.

DNA does not store the information to synthesize which of the following?

Organelles

Translation in eukaryotes

Primary transcripts are processed in the nucleus to produce a mature mRNA, which is then exported to the cytoplasm. Transcription and translation are separated in time and space. Once mRNAs are outside the nucleus, ribosomes can attach to them and begin translation. Polyribosomes form. Figure 17.9 a pg325

Proteins are synthesized at ribosomes and then released

Pulse-chase experiment

Elongation

RNA polymerase interior amino acids forms a rudder to help steer the template and non-template strands through channels inside the enzyme and the active site catalyzes the addition of nucleotides to the 3' end of the growing RNA molecule, the zipper helps separate the newly synthesized RNA from the DNA template. Enzyme's structure is critical for function, all prominent channels and grooves of the enzyme are filled.

Which of the following does not occur in post-transcriptional modifications occuring in eukaryotic mRNAs?

RNA polymerase termination

RNA polymerases

Responsible for synthesizing mRNA, perform a template-directed synthesis in the 5'--->3' direction, do not require a primer to begin transcription

Translation in bacteria

Ribosomes attach to mRNAs and begin synthesizing proteins even before transcription is complete. In fact, multiple ribosomes attach to each mRNA, forming a polyribosome. Many copies of a protein can be produced from a single mRNA. Transcription and translation can occur concurrently in bacteria because there is no nuclear envelope to separate the two processes.

Promoters

Sections of DNA that promote the start of transcription

Introns

Sections of primary transcript not in mRNA, because they are INTervening, not represented in the final RNA product

Transcription Description

Synthesis of RNA from a DNA template, formation of a phosphodiester linkage between ribonucleotides. RNA polymerase produces an RNA strand whose sequence is complementary to the bases in the DNA template strand. Once the holoenzyme is bound to a promoter for a bacterial gene, the DNA helix is opened by RNA polymerase, creating two separated strands of DNA When an incoming NTP pairs with a complementary base on the template strand of DNA, RNA polymerization begins. The reaction catalyzed by RNA polymerase is exergonic and spontaneous because NTPs have significant potential energy, owing to their three phosphate groups. Initiation ends as RNA polymerase extends the mRNA from the +1 site.

How is a mutation in a bacterial cell that deletes three base pairs 10 base pairs upstream from the +1 site likely to affect transcription and why?

The +1 site is the transcription start site and the region 10 base pairs upstream is a region of the promoter critical for sigma binding and initiation. Initiation will be inhibited because sigma cannot bind to the promoter.

An experimenter has altered the 3' end of the tRNA corresponding to the amino acid methionine in such a way as to remove the 3' AC. Which of the following hypotheses describes the most likely result?

The amino acid methionine will not bind.

Nonsense Mutation

The effect of a single base substitution depends on the new codon formed by the substitution. To identify the new codon, it is first necessary to determine the reading frame for the amino acid sequence. The first codon starts with base 1, the second codon with base 4, the third with base 7, and so on. In this problem, the codon that contains the single base substitution begins with base 34. The original codon (UUA, which encodes the amino acid leucine) is converted by the single base substitution to UAA, which is a stop codon. This will cause premature termination of translation, also called a nonsense mutation.

Sigma

The holoenzyme binds only to specific sections of DNA when sigma is present Binding sites are called promoters Responsible for guiding RNA polymerase to specific locations where transcription should begin

In eukaryotes...

The initial product is termed a primary transcript. This RNA must undergo multistep processing before it is functional. For protein-coding genes this primary transcript is called a pre-mRNA.

Ribosome binding site or Shine-Dalgarno sequence

The mRNA region in bacteria where a section of rRNA in a small ribosomal subunit binds to

In an experimental situation, a student researcher inserts an mRNA molecule into a eukaryotic cell after he has removed its 5' cap and poly-A tail. Which of the following would you expect him to find?

The molecule is digested by exonucleases since it is no longer protected at the 3' end.

+1 site

The place where transcription begins

Which of the following statements best describes the promoter of a protein-coding gene?

The promoter is the regulatory region of a protein-coding gene at which RNA polymerase must bind to initiate transcription—it is not transcribed into the RNA.

How does the bacterial ribosome recognize where to start translation?

The small ribosomal subunit binds to a sequence in the mRNA just upstream of the start codon

Ribosomes

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 tRNAs amino acid and the growing polypeptide chain. These events take place inside a ribosome. Contain rRNAs - ribosomal RNAs. Large subunit - where the peptide-bond formation takes place, small subunit - holds the mRNA in place during translation. A site - for Acceptor P site - holds the growing polypeptide chain, Peptidyl E site- for Exit 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 he 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. Occurs 20 times per second in bacterial ribosomes and 2 times per second in eukaryotic ribosomes. Starts at N-terminus and proceeds to C-terminus.

Transcription Rules

There are three principles to keep in mind when predicting the sequence of the mRNA produced by transcription of a particular DNA sequence. The RNA polymerase reads the sequence of DNA bases from only one of the two strands of DNA: the template strand. The RNA polymerase reads the code from the template strand in the 3' to 5' direction and thus produces the mRNA strand in the 5' to 3' direction. In RNA, the base uracil (U) replaces the DNA base thymine (T). Thus the base-pairing rules in transcription are A→U, T→A, C→G, and G→C, where the first base is the coding base in the template strand of the DNA and the second base is the base that is added to the growing mRNA strand.

Protein Pathways

There are two general targeting pathways for nuclear-encoded proteins in eukaryotic cells. Proteins that will ultimately function in the cytoplasm (PFK, for example) are translated on free cytoplasmic ribosomes and released directly into the cytoplasm. Proteins that are destined for the membranes or compartments of the endomembrane system, as well as proteins that will be secreted from the cell (insulin, for example), are translated on ribosomes that are bound to the rough ER. For proteins translated on rough ER, the proteins are found in one of two places at the end of translation. If a protein is targeted to a membrane of the endomembrane system, it will be in the ER membrane. If a protein is targeted to the interior of an organelle in the endomembrane system or to the exterior of the cell, it will be in the lumen of the rough ER. From the rough ER (membrane or lumen), these non-cytoplasmic proteins move to the Golgi apparatus for processing and sorting before being sent to their final destinations.

David Pribnow studied the base sequences of promoters in bacteria and bacterial viruses. He found two conserved regions in these promoters (the -10 box and the -35 box). What is the function of these two regions of the promoter?

They bind the sigma subunit that is associated with RNA polymerase.

tRNA

Transfer RNA, amino acids are transferred from the RNA to a growing polypeptide. The experiment also confirmed that tRNAs act as the interpreter during translation: tRNAs are Crick's adapter molecules. Serve as a chemical go-betweens that allow amino acids to interact with an mRNA template. Ranging from 75 to 85 nucleotides in length. Stem and loop structures. Stems - short stretches of double-stranded RNA. Loops - single stranded. Secondary structure: 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 of the structure could serve as an anticodon. Tertiary structure: tRNAs fold into an upside-down L-shaped molecule - anticodon at one end, CCAA sequence and attached amino acid at the other end. Vary at the anticodon and attached amino acid. It maintains a precise physical distance between the anticodon and the attached amino acid, which is important in positioning the amino acid and the anticodon within the ribosome. Figure 17.12 pg327

Elongation: Extending the polypeptide

When both the P site and A site are occupied by the tRNAs, the amino acids on the tRNAs are in the ribosome's active site This is where peptide bond formation - the essence of protein synthesis - occurs Figure 17.16 pg330 1. arrival of aminoacyl tRNA 2. peptide-bond formation 3. translocation - repeat down the length of the RNA

Release factor

When the translocating ribosome reaches one of the stop codons, this protein recognizes the stop codon and fills the A site

In bacteria...

When transcription terminates, the result is a mature mRNA that's ready to be translated into a protein.

Holoenzyme

Whole enzyme, consisting of a core enzyme which contains the active site for catalysis and other required proteins

You want to engineer a eukaryotic gene into a bacterial cell and have the gene expressed. What must be included in addition to the coding exons of the gene?

a bacterial promoter sequence

5' caps and 3' poly(A) tails of eukaryotic mRNAs ______.

protect mRNA from degradation and enhance translation

A mutant bacterial cell has a defective aminoacyl synthetase that attaches a lysine to tRNAs with the anticodon AAA instead of the normal phenylalanine. The consequence of this for the cell will be that

proteins in the cell will include lysine instead of phenylalanine at amino acid positions specified by the codon UUU.

During mRNA splicing ______.

snRNPs that make up the spliceosome recognize and remove introns

Where is an amino acid attached to a tRNA?

the 3' end

For any given gene, what ultimately determines which DNA strand serves as the template strand?

the base sequence of the gene's promoter In eukaryotes, binding of RNA polymerase II to DNA involves several other proteins known as transcription factors. Many of these transcription factors bind to the DNA in the promoter region (shown below in green), located at the 3' end of the sequence on the template strand. Although some transcription factors bind to both strands of the DNA, others bind specifically to only one of the strands. Transcription factors do not bind randomly to the DNA. Information about where each transcription factor binds originates in the base sequence to which each transcription factor binds. The positioning of the transcription factors in the promoter region determines how the RNA polymerase II binds to the DNA and in which direction transcription will occur.