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.
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.
DNA does not store the information to synthesize which of the following?
Organelles
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.
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.
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.
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.
Th
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),
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 t
During mRNA splicing ______.
snRNPs that make up the spliceosome recognize and remove introns
5' caps and 3' poly(A) tails of eukaryotic mRNAs ______.
protect mRNA from degradation and enhance translation
What is recognized by an aminoacyl tRNA synthetase?
one amino acid and the set of tRNAs that are coupled to that amino acid
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
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
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.
Pr
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
Why does cordycepin end transcription?
It lacks a 3' OH.
Where is an amino acid attached to a tRNA?
the 3' end
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.
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.
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.
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'
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.
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.
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.
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
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
NTP
Has a hydroxyl (OH-) group on the 2' carbon (ribose instead of deoxyribose)
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)
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
Template Strand
Strand that is read by the enzyme
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)
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
Eukaryotes
At least three types of RNA polymerase
Initiation
Sigma must bind to the polymerase before transcription can begin, bacterial RNA polymerase and sigma form a holoenzyme
Phases of Transcription
Initiation, elongation, termination
Holoenzyme
Whole enzyme, consisting of a core enzyme which contains the active site for catalysis and other required proteins
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
Promoters
Sections of DNA that promote the start of transcription
-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
Downstream
DNA located in the direction RNA polymerase moves during transcription
Upstream
DNA located in the opposite direction that RNA polymerase moves during transcription
+1 site
The place where transcription begins
-35 box
TTGACA 35 bases upstream from the +1 site
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
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 shor
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 m
In bacteria...
When transcription terminates, the result is a mature mRNA that's ready to be translated into a protein.
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.
Discovery of Introns
Loops in the micrograph representing regions of DNA that are transcribed but are not found in the final mRNA
Exons
Part of the final mRNA, because they at EXpressed
Introns
Sections of primary transcript not in mRNA, because they are INTervening, not represented in the final RNA product
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 transcript
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
RNA processing
Any of the modifications needed to convert a primary transcript into a mature RNA
Proteins are synthesized at ribosomes and then released
Pulse-chase experiment
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 transl
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 t
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 acid
Anticodon
A set of three ribonucleotides that forms base pairs with the mRNA codon.
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, ther
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
Aminoacyl tRNA
The combination of a tRNA molecule covalently linked to an 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
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 insi
Ribosome binding site
or Shine-Dalgarno sequence
The mRNA region in bacteria where a section of rRNA in a small ribosomal subunit binds to
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
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
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. p
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
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 th
Release factor
When the translocating ribosome reaches one of the stop codons, this protein recognizes the stop codon and fills the A site
Molecular Chaperones
Speed up folding of proteins