CH11 The Central Dogma: DNA to RNA to Protein (karp)

gene expression

refers to the production of a functional product (for example, an enzyme) using the information encoded in a gene. The information present in a segment of DNA is made available to the cell through formation of a molecule of RNA.

Transcription

process in which the information encoded in the four deoxyribonucleotide letters of DNA is rewritten, or transcribed, into a similar language composed of four ribonucleotide letters of RNA

messenger RNA (mRNA)

intermediate between a gene and its polypeptide; can also be described as a "sense" strand or a coding RNA

allows the cell to separate information storage from information utilization and allows a cell to greatly amplify its synthetic output.

Once in the cytoplasm, it can serve as a template to direct the incorporation of amino acids in a particular order encoded by the nucleotide sequence of the DNA and mRNA.

Central Dogma

Concept of a DNA-based gene encoding an RNA‐based message that is then translated into a protein is known as

Translation

Proteins are synthesized in the cytoplasm by a complex process called?

It requires the participation of dozens of different components, including ribosomes.

Ribosomes

Complex, cytoplamic "machines" that can be programmed, like a com- puter, to translate the information encoded by any mRNA and it also contains both protein and RNA.

ribonucleoprotein machines responsible for protein synthesis

ribosomal RNAs (rRNAs)

provide structural support to build the ribosome and help to catalyze the chemical reaction in which amino acids are covalently linked to one another.

80 percent of the RNA in most cells consists of this

play major roles in selecting tRNAs, ensuring accurate translation, binding protein factors, and polymerizing amino acids

Transfer RNAs (tRNAs)

are required to translate the information in the mRNA nucleotide code into the amino acid "alphabet" of a polypeptide.

constitute a third major class of RNA that is required during protein synthesis.

TRUE

PROTEIN FOLDING is driven by the withdrawal of hydrophobic residues into the interior, RNA FOLDING is driven by the formation of regions having complementary base pairs. (TRUE OR FALSE)

DNA‐dependent RNA polymerases (RNA polymerases or RNAP)

These enzymes are able to incorporate nucleotides, one at a time, into a strand of RNA whose sequence is complementary to one of the DNA strands, which serves as the template.

promoter

The site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription

transcription factors

Collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription.

RNA polymerases are not capable of recognizing promoters on their own but require the help of additional pro- teins called

RNA(n) + NTP --> RNA(n+1) + PPi

RNA polymerase catalyzes the highly favorable reaction, in which ribonucleoside triphosphate substrates (NTPs) are cleaved into nucleoside monophosphates as they are polymerized into a covalent chain

PPi --> 2Pi

a second favorable reaction catalyzed by a different enzyme, a PYROPHOSPHATASE. In this case, the pyrophosphate (PPi) produced in the first reaction is hydrolyzed to inorganic phosphate (Pi). The hydrolysis of pyrophosphate releases a large amount of free energy and makes the nucleotide incorporation reaction essentially irreversible.

True

Even though polymerases are relatively powerful motors, these enzymes do not necessarily move in a steady, continuous fashion but may pause at certain locations along the template or even backtrack before resuming their forward progress (True or False)

Proofreading

ability of RNA polymerase to recognize and remove a misincorporated nucleotide is referred to as?

This corrective function is carried out by the same active site within the enzyme that is responsible for nucleotide incorporation.

Topoisomerase

corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands

core enzyme (bacteria)

a single type of RNA polymerase composed of five subunits that are tightly associated to form a?

sigma factor (σ)

A bacterial protein that, as part of the RNA polymerase holoenzyme, recognizes the -10 and -35 regions of bacterial promoters, thus positioning the holoenzyme to initiate transcription correctly at the start site. The _____ dissociates from the holoenzyme before RNA synthesis.

Attachment of ____ to the core enzyme increases the enzyme's affinity for promoter sites in DNA and decreases its affinity for DNA in general.

Holoenzyme

RNA polymerase + sigma factor

open complex

The complex of the polymerase, σ factor, and DNA with the strands separated is called the

transcriptional elongation complex

Once about 10 nucleotides have been successfully incorporated into a growing transcript, the enzyme undergoes a major change in conformation and is transformed into a _____________ that can move processively along the DNA.

formation of an ________ is followed by release of the σ factor.

consensus sequence

The conserved sequence that is approximately 35 bases upstream from the initiation site; TTGACA sequence (known as the −35 element) is called a?

Pribnow box

second conserved sequence is found approximately 10 bases upstream from the initiation site and occurs at the consensus sequence TATAAT;

This site in the promoter, named (the -10 element) after its position or the?

sigma factor 70

known as the "housekeeping" factor, because it initiates transcription of most genes.

heat-shock genes

The products of these genes protect the proteins of the cell from thermal damage.

Rho

ring‐shaped protein called _____ is required for termination of bacterial transcription.

It encircles the newly synthesized RNA and moves along the strand in a 5′→3′ direction to the polymerase, where it separates the RNA transcript from the DNA to which it is bound.

terminator sequence

polymerase stops transcription when it reaches a?

typically fold into a hairpin loop that causes the RNA polymerase to release the completed RNA chain without requiring additional factors.

Transcription factors

These proteins play a role in virtually every aspect of the transcription process, from the binding of the polymerase to the DNA template, to the initiation of transcription, to its elongation and termination

primary transcript (pre-RNA)

initial precursor RNA is equivalent in length to the full length of the DNA transcribed and is called

Transcription unit

The corresponding segment of DNA from which a primary transcript is transcribed is called

mRNAs, rRNAs, and tRNAs

derived from precursor RNA molecules that are considerably longer than the final RNA product.

rDNA

To furnish the cell with such a large number of transcripts, the DNA sequences encoding rRNA are normally repeated hundreds of times. This DNA, called

typically clustered in one or a few regions of the genome. The human genome has five __________ clusters, each on a different chromosome.

nucleoli (singular: nucleolus)

nondividing (interphase) cell, the clus- ters of rDNA are gathered together as part of one or more irregularly shaped nuclear structures, called_________, that function in producing ribosomes

28S, 5.8S, and 5S RNA

Large subunit of Eukaryotic Ribosomes contains these three rRNA

18S RNA

Small subunit of Eukaryotic Ribosomes contains this one rRNA molecule

RNA polymerase I

Enzyme that synthesize larger rRNAs (28S, 18S, 5.8S) and is specialized for transcribing the pre‐rRNA

RNA polymerase III

5S rRNA is synthesized from a sepa- rate RNA precursor outside the nucleolus by a different enzyme called?

It also synthesizes other small RNAs, including tRNAs, 5S rRNA, and U6 snRNA

45S RNA

This is cleaved into smaller molecules that are then trimmed down to the 28S, 18S, and 5.8S rRNA molecules.

32S RNA

a precursor to the mature 28S and 5.8S rRNAs

mature 18S rRNA

other major product of the 45S pre‐rRNA leaves the nucleolus quite rapidly and appears in the cytoplasm as the __________

small nucleolar RNAs (snoRNAs)

processing of the pre‐rRNA is accomplished with the help of a large number of ________ ___________ ___________?

small, nucleolar ribonucleoproteins (or snoRNPs)

small, nucleolar RNAs (or snoRNAs) that are packaged with particular proteins to form particles called

box C/D snoRNAs

Low abundance snoRNAs that determine which nucleotides in the pre‐rRNA will have their ribose moieties methylated

box H/ACA snoRNAs

Low abundance snoRNAs that determine which uridines are converted to pseudouridines. The structures of nucleotides modified by these two reactions

True

snoRNAs provide an excellent example of the principle that single‐stranded nucleic acids having complementary nucleotide sequences are capable of forming double‐stranded hybrids. In this case, each snoRNA binds to a specific portion of the pre‐rRNA to form an RNA-RNA duplex. (TRUE or FALSE)

either a methylase or a pseudouridylase

The bound snoRNA then guides an enzyme (what enzyme?) within the snoRNP to modify a particular nucleotide in the pre‐rRNA.

RNA helicases

enzymes that unwind regions of double‐stranded RNA

True

The nucleolus is the site not only of rRNA processing, but also of assembly of the two ribosomal subunits (true or false)

5s rRNA

These RNA molecules are encoded by a large number of identical genes that are separate from the other rRNA genes and are located outside the nucleolus. Following its synthesis, the ________________ is transported to the nucleolus to join the other components involved in the assembly of ribosomal subunits.

Synthesized by the RNA polymerase III

dyskerin

The most frequent modifications to nucleotides in a pre‐rRNA are the conversion (isomerization) of a uridine to a pseudouridine and the methylation of a ribose at the 2′ site of the sugar. To convert uridine to pseudouridine, the N1 C1′ bond is cleaved, and the uracil ring is rotated through 120°, which brings the C5 of the ring into place to form the new bond with C1′ of ribose.

These chemical modifications are catalyzed by a protein component of the snoRNPs called?

tDNA

The DNA within a cluster consists largely of nontranscribed spacer sequences with the tRNA coding sequences situated at irregular intervals in a tandemly repeated arrangement

RNA polymerase III

tRNAs are transcribed by the enzyme?

True

The promoter sequence of tRNA genes lies within the coding section of the gene rather than being located at its 5′ flank. (True or false)

ribonuclease P

One of the enzymes involved in pre‐tRNA processing is an endonuclease called _______________ , which is present in both bacterial and eukaryotic cells and consists of both RNA and protein subunits.

It is the RNA subunit of _____________ that catalyzes cleavage of the pre‐tRNA substrate

general transcription factors

Transcription of RNA begins when RNAP binds to the DNA at a region called the promoter. The process is facilitated by a group of proteins called?

required for accurate initiation of tran- scription of a diverse array of genes in a wide variety of different organisms.

heterogeneous nuclear RNAs (hnRNAs)

When eukaryotic cells are incubated for a short period in [3H] uri-

dine or [32P] phosphate and immediately killed, most of the radioactivity is incorporated into RNA molecules that have the following properties: (1) they have large molecular weights (up to about 80S, or 50,000 nucleotides); (2) as a group, they are represented by RNAs

of diverse (heterogeneous) nucleotide sequence; and (3) they are found only in the nucleus. Because of these properties, these RNAs are referred to as?

primarily precursors to the

smaller cytoplasmic mRNAs; these are processed into mRNAs (or completely degraded) even as they are being synthesized.

general transcription factors (GTFs); preinitiation complex (PIC)

RNA polymerase II binds the promoter with the cooperation of a number of __________ to form a ___________.

Initiator (Inr)

Many eukaryotic promoters contain a second, conserved core promoter element called the _________, which includes the site where transcription is initiated

TBP‐associated factors (TAFs)

TFIID includes the TBP subunit, which specifically binds to the TATA box, and a number of other subunits, which collectively are called?

TFIIF

A general transcription factor which contains a subunit homologous to the bacterial σ factor, is bound to the entering polymerase.

TATA‐binding protein (TBP)

The first step in assembly of the preinitiation complex is bind- ing of a protein, called_______, that recognizes the TATA box of these promoters

It is present as a subunit of a much larger protein complex called TFIID

(Is also the subunits of SL1 or TFIIIB, which promotes the binding of RNA polymerases I and III, respectively.)

TFIID

While TBP binds TATA, other subunits of the _________ complex bind to other regions of the DNA, including elements that lie downstream of the transcription start site.

Binding of_______ sets the stage for the assembly of the complete preinitiation complex

TBP of TFIID, TFIIA, and TFIIB;

TFIIE and TFIIH

Interaction of three of the GTFs bound (name them?) to the promoter provides a platform for the subsequent binding of the huge, multi- subunit RNA polymerase with its attached TFIIF. Once the RNA polymerase-TFIIF is in position, another pair of GTFs (name the two?) join the complex and transform the polymerase into an active, transcribing machine.

TFIIH

only GTF known to possess enzymatic activities?

One of the subunits of ______ functions as a protein kinase to phos-phorylate RNA polymerase, whereas two other subunits of this protein complex function as DNA unwinding enzymes (helicases).

True

Once transcription begins, certain of the GTFs (including TFIID) may be left behind at the promoter, while others are released from the complex.

As long as TFIID remains bound to the promoter, additional RNA polymerase molecules may be able to attach to the promoter site and rapidly initiate additional rounds of transcription. (TRUE OR FALSE)

carboxyl‐terminal domain (CTD)

It is the largest subunit of RNA polymerase II that has an unusual structure; it consists of a sequence of seven amino acids (‐Tyr1‐Ser2‐Pro3‐Thr4‐Ser5‐Pro6‐Ser7‐) that is repeated over and over. In humans, the ______ consists of 52 repeats of this heptapeptide.

Also acts as a platform for the dynamic gain and loss of factors required for the formation of a mature mRNA.

Phosphorylation by TFIIH

may act as the trigger that uncouples the enzyme from the GTFs and/or the promoter DNA, allowing the enzyme to escape from the preinitiation complex and move down the DNA template.

P‐TEFb

another kinase that phosphorylates the CTD on the serine residues at position #2. This change in phosphorylation pattern is thought to facilitate the recruitment of

additional protein factors involved in RNA splicing and addition of a poly(A) tail

Intervening Sequences (introns)

regions of DNA between these blocks, referred to as ______ and are somehow missing in the corresponding mRNA.

split genes

Intervening sequences were soon found in other genes, and it became apparent that the presence of genes with intervening sequences—called ___________ ____________—is the rule not the exception.

Exons

The parts of a split gene that contribute to the mature RNA prod- uct are called

5′ cap and 3′ poly(A) tail

The conversion process of pre-mRNA to mature mRNA requires addition of a __________ and ___________ to the ends of the transcript, and removal of any intervening introns.

methylguanosine cap

It prevents the 5′ end of the mRNA from being digested by exonucleases, it aids in transport of the mRNA out of the nucleus, and it plays an important role in the initiation of mRNA translation.

poly(A) tail

The 3′ end of an mRNA contains a string of adenosine residues form a ________. This sequence, AAUAAA, in the primary transcript serves as a recognition site for the assembly of a large complex of proteins that carry out the processing reactions at the 3′ end of the mRNA.

This together with an associated protein protects the mRNA from premature degradation by exonu- cleases.

poly(A) polymerase

This enzyme adds 250 or so adenosines without the need of a template

RNA splicing

The parts of a primary transcript that correspond to the intervening DNA sequences (the introns) must be removed by a complex process known as?

Ligation of exons

allows the cut ends of the exon to be covalently joined

exon junction complex (EJC)

a protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization, and degradation; stays with the mRNA until it is translated

the 5′ splice site, the 3′ splice site, and the polypyrimidine tract

The sequence most commonly found at the exon-intron borders within mammalian pre‐mRNA molecules. The G/GU at the 5′ end of the intron (?), the AG/G at the 3′ end of the intron (?), and the _______________ near the 3′ splice site are present in the vast majority of eukaryotic pre‐mRNAs

preferred nucleotides

In addition, the adjacent regions of the intron contain _______________ ____________, which play an important role in splice site recognition.

exonic splicing enhancers (ESE)

The additional clues that allow the splicing machinery to distinguish between exons and introns are provided by specific sequences, most notably the ones situated within exons

Seq. Situated within exons that play a key role in recognition of exons by the splicing machinery. They serve as binding sites for a family of RNA‐binding proteins, called SR proteins

SR proteins

Group of serine- and arginine-rich proteins that regulate alternative splicing of pre-mRNA.

ESEs serves as binding sites for this family of RNA-binding proteins

They are thought to form interacting networks that span the intron/exon borders and help recruit snRNPs to the splice sites. Positively charged _____________ may also bind electrostatically to the negatively charged phosphate groups that are added to the CTD of the polymerase as transcription is initiated

ribozymes

RNA enzymes

lariat (bacterial)

Group II introns undergo self‐splicing by passing through an intermediate stage, called

The formation of this is by a covalent bond between the 5′ end of the intron and an adenosine residue near the 3′ end of the intron

True

pre‐mRNA from eukaryotic cells is not able to splice itself as compared to its bacterial counterpart (true or false)

small nuclear RNAs (snRNAs) and their associated proteins

This helps splice pre-mRNA because eukaryotic cells cannot splice pre-mRNA by itself and requires instead a host of?

heterogeneous nuclear ribonucleoprotein (hnRNP)

represents the substrate for the processing reaction of mRNAs

spliceosome

Processing occurs as each intron of the pre‐mRNA becomes associated with a dynamic macromolecular machine called a _____________.

Each __________ consists of a variety of proteins and a number of distinct ribonucleoprotein particles, called small nuclear ribonucleoproteins (snRNPs) because they are composed of snRNAs bound to specific proteins.

These are not present within the nucleus in a prefabricated state, but rather are assembled as their component snRNPs bind to the pre‐mRNA.

small nuclear ribonucleoproteins (snRNPs)

Each spliceosome consists of a variety of proteins and a number of distinct ribonucleoprotein particles, called _____________ because they are composed of snRNAs bound to specific proteins.

carry out the reactions that cut the introns out of the transcript and paste the ends of the exons together.

Sm proteins

One family, called ____________, are present in all of the snRNPs. These bind to one another and to a conserved site on each snRNA (except U6 snRNA) to form the core of the snRNP.

ATP‐consuming RNA helicase

The multiple rearrangements among RNA mole- cules that occur during the assembly of a spliceosome are primarily mediated by _________, present within the snRNPs.

True

(1) pre‐mRNAs are spliced by the same pair of chemical reactions that occur as group II introns splice themselves and (2) the snRNAs that are required for splicing pre‐mRNAs closely resemble parts of the group II introns suggested that the snRNAs are the catalytically active components of the snRNPs, not the proteins.

According to this scenario, the spliceosome would act as a ribozyme and the proteins would serve various supplementary roles, such as maintaining the proper three‐dimensional structure of the snRNA and selecting the splice sites to be used during the processing of a par- ticular pre‐mRNA.

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins.

Many primary transcripts can be processed by two or more pathways so that a sequence that acts as an intron in one pathway becomes an exon in an alternate pathway;

same gene can code for more than one polypeptide.

exon shuffling

Mechanism for the evolution of new genes; in the process, coding sequences from different genes are brought together to generate a protein with a novel combination of domains.

The movement of genetic "modules" among unrelated genes—a process called __________ —is greatly facilitated by the presence of introns, which act like inert spacer elements between exons. Genetic rearrangements require breaks in DNA molecules, which can occur within introns without introducing mutations that might impair the organism. Over time, exons can be shuffled independently in vari- ous ways, allowing a nearly infinite number of combinations in search for new and useful coding sequences.

types of reactions that RNA molecules are capable of catalyzing

cleavage and ligation of phosphodiester bonds required for RNA splicing and the formation of peptide bonds during protein synthesis are?

a RIBONUCLEOTIDE REDUCTASE to convert ribonucleotides into deoxyribonucleotides and a REVERSE TRANSCRIPTASE to transcribe RNA into DNA.

The evolution of DNA may have required only two types of enzymes

posttranscriptional gene silencing (PTGS)

A phenomenon where added genes and their normal counterparts within the genome were being transcribed, but the resulting mRNAs were somehow degraded.

RNA interference (RNAi)

One of the preparations contained "sense" RNA, that is, an RNA having the sequence of the mRNA that encoded the protein being targeted; another preparation contained "antisense" RNA, that is, an RNA having the complementary sequence of the mRNA in question; and a third preparation consisted of a double‐stranded RNA containing both the sense and antisense sequences bound to one another.

The phenomenon where neither of the single‐stranded RNAs had much of an effect, but the double‐stranded RNA was very effective in stopping production of the encoded protein is called?

Is thought to have evolved as a type of "genetic immune system" to protect organisms from the presence of foreign or unwanted genetic mate- rial. To be more specific, __________ probably evolved as a mechanism to block the replication of viruses and/or to suppress the movements of transposons within the genome because both of these potentially dangerous processes can involve the formation of double‐stranded RNAs. Cells can recognize dsRNAs as "undesirable" because such molecules are not produced by the cell's normal genetic activities.

"sense" RNA

An RNA having the sequence of the mRNA that encoded the protein being targeted