What is the function of the release factor during translation in eukaryotes?

Translation Termination and mRNA Stability

Eukaryotic mRNA translation termination requires two release factors, eRF1 and eRF3. Translation termination process can influence mRNA half-life. Specifically, it was observed that the N-terminal domain of eRF3, which is not required for translation termination, can interact with Pab1, and this interaction is involved in modulating mRNA stability (Hosoda et al., 2003). Disruption of this interaction results in translation-dependent stabilization of mRNA caused by decreased deadenylation rate (Hosoda et al., 2003). Interestingly, it was further found that certain deadenylase complexes can also bind to the same site on Pab1 that is involved in the interaction with eRF3 (Funakoshi et al., 2007). Thus, it has been postulated that eRF3 can regulate mRNA deadenylation by competitively binding to the Pab1, which then modulates the recruitment and activation of deadenylase complexes (Funakoshi et al., 2007). In addition to the release factors, other proteins that can modulate translation termination can also influence mRNA stability. For example, a recent characterized protein named Tpa1 can interact with the two release factors and regulate the readthrough of stop codons (Keeling et al., 2006). Interestingly, although the detailed mechanisms still remain elusive, knocking out this protein can have decreased deadenylation rate and increased mRNA stability (Keeling et al., 2006). Collectively, these results suggest that mRNA translation termination can results in mRNP conformational changes that can influence mRNA stability, likely via modulating mRNA deadenylation.

Release Factors

Prokaryotic Polypeptide Chain Release Factors

In bacteria, translation termination is controlled by three different RFs (Table I). Two class-I protein release factors, RF1 and RF2, each decode two of three stop codons, UAA or UAG (RF1) and UAA or UGA (RF2). Recognition of the stop codon by the RFs is mediated via a conserved tripeptide motif: Pro-Ala-Thr (PAT) in RF1 and Ser-Pro-Phe (SPF) in RF2. These tripeptides are referred to as peptide anticodons. The other important functional domain of class-I RFs contains the highly conserved amino acid motif Gly-Gly-Gln (GGQ) and it is this domain that triggers hydrolysis of the protein-tRNA bond. An understanding of how RFs trigger the release of the completed polypeptide from the tRNA at the P site has come from a study of RF2 and its interaction with the terminating ribosome. When RF2 binds to the ribosome with a stop codon positioned at the A site, RF2 changes its three-dimensional conformation such that the domain with the conserved “peptide anticodon” (SPF) interacts with the mRNA at the decoding center, and the GGQ-containing domain comes in the contact with the ribosomal PTC to trigger hydrolysis of the peptidyl-tRNA linkage (Figure 2).

Figure 2. The three-dimensional conformation of a release factor changes once it is bound to the ribosome. (A) The RF2:bacterial ribosome complex indicating the points of contact between RF2 and the decoding center and the PTC of the ribosome. (B) The three-dimensional structures of the unbound and bound forms of bacterial RF2 indicating the location of the “peptide anticodon” sequence Ser-Pro-Phe (SPF) and the conserved Gly-Gly-Gln (GGQ) amino acid motif.

The single class-II RF in bacteria, namely RF3, is a GTP-binding protein that accelerates dissociation of either RF1 or RF2 from the ribosomal A-site after release of the completed polypeptide chain from the ribosome. RF3 bound to GDP accesses the ribosome which, in complex with RF1 or RF2, acts as guanine nucleotide exchange factors (GEFs) and triggers dissociation of GDP from RF3. This leads to the formation of a stable ribosome-RF1 (or RF2-) RF3 complex. Hydrolysis of the peptidyl-tRNA linkage triggered by RF1 or RF2 allows GTP binding to RF3. This induces an altered RF3 conformation with a high affinity for the ribosome and leads to rapid dissociation of RF1 or RF2 from the termination complex. To leave the ribosome, RF3 requires GTP hydrolysis, which converts it to the GDP-bound form of RF3 which has a lower affinity for the ribosome. Once RF3 leaves the ribosome, it is ready to enter the next translation cycle.

Eukaryotic Chain Release Factors

In contrast to bacteria, in eukaryotic cells translation is terminated by a single heterodimer consisting of two different RFs, eRF1 and eRF3. eRF1 is a class-I RF that decodes all three stop codons and triggers peptidyl-tRNA hydrolysis by the ribosome to release the nascent polypeptide. In eRF1, the stop codon recognition site is located close to the amino terminus of the protein molecule in a region that contains an evolutionarily conserved tetrapeptide sequence, Asn-Ile-Lys-Ser (NIKS). This sequence may be functionally equivalent to the bacterial RF peptide anticodon. The GGQ motif found in bacterial RF1 and RF2 is located in a separate domain of the protein to the NIKS sequence. The carboxy-terminal part of the eRF1 binds eRF3. The crystal structure of human eRF1 shows that it is a Y-shaped molecule that resembles the structure of a tRNA (Figure 3). Since both bacterial and eukaryotic class-I RFs carry out essentially the same function, one would expect them to interact similarly with ribosomal A site. As with RF2 (see Figure 2), eRF1 must also undergo a conformational change after binding to the ribosome in order that it can interact with both the ribosomal decoding site and the ribosomal PTC.

Figure 3. Many of the protein factors involved in translation elongation and termination have three-dimensional shapes that mimic a tRNA molecule. (A) Three-dimensional structure of a tRNA showing the location of the anticodon and the site to which the amino acid is covalently attached. (B) The three-dimensional structures of the mammalian RF eRF1 (left) and the bacterial translation elongation factor EF-G (right). The position of the conserved NIKS and GGQ amino acid motifs are indicated on eRF1, together with the domain to which the class-II factor eRF3 binds.

The eukaryotic class-II RF, eRF3, forms a complex with eRF1, and via this interaction enhances the efficiency of the translation termination although its function remains to be fully defined. The GTPase activity of eRF3, which is triggered by stop codons, is both eRF1- and ribosome dependent. The carboxy-terminal half of the eRF3 molecule shows significant amino acid identity to the translation elongation factor eEF1A that is responsible for bringing the aminoacyl tRNAs to the ribosome during polypeptide chain elongation. This suggests that eRF3 may act in a manner analogous to eEF1A, a protein factor bringing the RF complex to the ribosome. A number of functions have been attributed to eRF3, e.g., it may displace eRF1 from the ribosome (i.e., the function assigned to prokaryotic RF3), or proofread the eRF1:stop codon interaction, or stimulate ribosome disassembly (i.e., the function assigned to prokaryotic RRF; see below), although direct experimental proof for any one specific function is lacking. In mammalian cells, eRF3 binds to the poly(A)-binding protein (PABP), a protein associated with the poly(A) tails located at the 3′ end of the majority of eukaryotic mRNAs. This interaction might be important for the regulation of both mRNA stability and translation initiation, since disruption of the eRF3::PABP interaction suppresses the recycling of the ribosome on the same mRNA.

In Bakers' yeast (Saccharomyces cerevisiae), the eRF3 protein (also known as Sup35p) has an additional remarkable property; it is a prion protein that acts as the protein determinant of a non-Mendelian genetic element called [PSI+]. Like the mammalian prion protein PrP, eRF3 can exist in one of two different conformations with the aggregation-prone prion conformer catalyzing the conversion of the normal soluble form, to the prion form. In [PSI+] cells the majority of eRF3 is found as an inactive, high molecular weight complex, and thus [PSI+] cells have a defect in translation termination albeit without detriment to yeast cell growth. This mechanism may represent a novel means of regulating the efficiency of translation termination in yeast. There is no evidence that mammalian eRF3 is a prion.

In Archaebacteria, the single class-I RF found (aRF1) shares significant amino acid sequence and structural homology with eRF1, but not with either RF1 or RF2. Like eRF1, aRF1 is able to decode all three stop codons. No eRF3 homologue has been identified in any Archaebacterial genome.

D. Termination

Since Chlamydomonas encodes close homologues of the eubacterial termination factors, translation termination in chloroplasts is likely to be mechanistically similar (reviewed in Ramakrishnan, 2002). Translation termination begins when a stop codon is encountered in the A-site of the ribosome. Two “class I” release factors, RF1 (which recognize UAA and UAG) and RF2 (which recognizes UAA) together with the “class II” release factor RF3, release the completed polypeptide. Binding of RF1/2 to a ribosome triggers the hydrolysis and release of the peptide chain from the tRNA in the P site. RF3 promotes rapid dissociation of RF1 and RF2. After release of the peptide chain, the ribosome is left with mRNA and a deacylated tRNA in the P site. Ribosome recycling factor (RRF) and EF-G are required to disassemble this complex (Table 28.4; Rolland et al., 1999).

In Arabidopsis, screens of albino and high chlorophyll fluorescence (hcf) mutants uncovered genes for both RRF1 and RF2 (Meurer et al., 2002; Motohashi et al., 2007). RRF1 was uncovered through a screen of albino or pale-green (apg) mutants, where apg3-1 carried a mutation in the chloroplast homologue AtcpRF1 (Motohashi et al., 2007). Complementation analysis using the E. coli rf1 mutant revealed that APG3 does function as an RF1 in E. coli. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is essential for chloroplast development.

The hcf109 mutation identified a chloroplast-targeted RF2-like protein (AtpfrB), whose absence causes decreased stability of UGA-containing mRNAs (Meurer et al., 2002). This implicates AtpfrB in the regulation of both mRNA stability and protein synthesis. Chlamydomonas may have lost this “auxiliary” function of RF2 as only two ORFs in the plastome encode UGA termination codons (Maul et al., 2002). If this is the case, reporter genes containing TGA stop codons would be predicted to not function because of a failure to terminate translation. There are two reports that have shown that UGA stop codons can be functional in Chlamydomonas chloroplasts (Lee et al., 1996; Sizova et al., 1996). However, in both of these reports, the possibility of frameshifting and readthrough was not investigated.

D trans-Acting Factors Involved in the Nonsense-Mediated mRNA Decay Pathway

Nonsense suppressors in yeast are either tRNA mutants, capable of decoding a translation termination codon, or mutants with lesions in non-tRNA genes, which enhance the expression of nonsense-containing alleles by other mechanisms. The latter mutants include the allosuppressors, frameshift suppressors, and omnipotent suppressors (Surguchov, 1988; Hinnebusch and Liebman, 1991). At least one of these suppressors, upf1, acts by suppressing nonsense-mediated mRNA decay.

Mutants in the UPF1 gene (and in UPF2, -3, and -4) were isolated on the basis of their ability to act as allosuppressors of the his4-38 frameshift mutation (Culbertson et al., 1980). The latter mutation is a single G insertion in the HIS4 gene that generates a + 1 frameshift and a UAA nonsense codon in the triplet adjacent to the insertion (Donahue et al., 1981). At 30°C, but not 37°C, his4-38 is suppressed by SUF1-1, which encodes a glycine tRNA capable of reading a four base codon (Mendenhall et al., 1987). Mutations in UPF1 allow cells that are his4-38 and SUF1-1 to grow at 37°C (Culbertson et al., 1980). The basis of this suppression appears to be the loss of function of a trans-acting factor (Upf1p) essential for nonsense-mediated mRNA decay. Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs (Table 2) (Leeds et al., 1991). Thus, in a UPF1 deletion mutant (upf1Δ), the his4-38 mRNA is stabilized approximately 5-fold, halflives of mRNAs from the PGK1 early nonsense alleles are stabilized approximately 12-fold, and half-lives of the PGK1 mRNAs with late nonsense codons or mRNAs from the wild-type MATαl, STE3, LEU2, HIS4, PGK1, PAB1, and ACT1 genes are unchanged (Table 2). Regardless of position, all of the PGK1 nonsense alleles have mRNA half-lives on the order of an hour in a upf1Δ strain (Peltz et al., 1993), a result that indicates that the loss of UPF1 function restores wild-type decay rates to mRNAs that would otherwise have been susceptible to the enhancement of decay rates promoted by early nonsense codons.

Suppression of nonsense-mediated mRNA decay in upf1Δ strains does not appear to result from enhanced read-through of the translation termination signal (Leeds et al., 1991), nor does it appear to be specific for a particular nonsense codon. The ability of upf1− mutants to suppress tyr7-1 (UAG), leu2-1 (UAA), leu2-2, (UGA), met8-1 (UAG), and his4-166 (UGA) (Leeds et al., 1992) indicates that they can act as omnipotent suppressors. Since many biosynthetic pathways do not require maximal levels of gene expression for cell survival (e.g., only 6% of the HIS4 gene product is required for growth on plates lacking histidine; Gaber and Culbertson, 1984), stabilization of nonsense-containing transcripts would allow synthesis of sufficient read-through protein to permit cells to grow on media lacking the relevant amino acids.

The UPF1 gene has been cloned and sequenced and shown to be (1) nonessential for viability, (2) capable of encoding a 109-kDa protein with both zinc finger and nucleotide (GTP) binding site motifs, and (3) partially homologous to the yeast SEN1 gene (Leeds et al., 1992). The latter encodes a noncatalytic subunit of the tRNA splicing endonuclease complex (Winey and Culbertson, 1988), suggesting that Upf1p may also be part of a nuclease complex targeted to nonsense-containing mRNAs. It is unlikely, however, that its normal function is anticipatory, i.e., that it is solely involved in the degradation of mRNAs with premature nonsense codons. One role of the UPF1 gene may be to regulate the decay rates of transcripts with upstream open reading frames, a conclusion that followed from an analysis of the steady-state levels and decay rates of mRNAs encoding gene products involved in the pyrimidine biosynthetic pathway (S. W. Peltz, A. H. Brown, J. Wood, A. Atkins, P. Leeds, M. Culbertson, and A. Jacobson, unpublished experiments). Transcription of several genes in this pathway (URA1, URA3, and URA4) is governed by the positive activator, PPR1 (Losson et al., 1983; Kammerer et al., 1984; Liljelund et al., 1984). The PPR1 mRNA itself has a small (five codon) open reading frame upstream of the PPR1 coding sequence; the termination codon for the upstream open reading frame overlaps with the ATG of the PPR1 coding sequence (Losson et al., 1983). Measurement of PPR1 mRNA decay rates demonstrates that it decays threefold more slowly in a upf1− strain than in a UPF1+ strain [t1/2 = 3 min (upf1−) vs t1/2 = 1 min (UPF1+); S. W. Peltz, A. H. Brown, J. Wood, A. Atkins, P. Leeds, M. Culbertson, and A. Jacobson, unpublished experiments], suggesting that upstream open reading frames, in addition to reducing the frequency of downstream translational initiation (Kozak, 1991; Hinnebusch and Liebman, 1991), may also destabilize specific transcripts. This destabilization mechanism, which would involve the UPF1 gene product, would not necessarily pertain to all mRNAs with upstream open reading frames since there is no effect of UPF1 status on the decay rate of the GCN4 mRNA (Table 2; S. W. Peltz, A. H. Brown, J. Wood, A. Atkins, P. Leeds, M. Culbertson, and A. Jacobson, unpublished experiments).

The possibility also exists that mRNAs with splicing errors constitute another class of UPF1 substrate; i.e., splicing fidelity would be maintained by the rapid turnover of RNAs in which processing errors have caused the insertion or deletion of one or more nucleotides. At present, there is no evidence to suggest an error-prone splicing process. However, a related phenomenon, premature transport of pre-mRNA from the nucleus, may occur (Yost and Lindquist, 1988; reviewed in Kozak, 1991). Translation of intron-containing RNAs would almost certainly lead to premature termination and, thus, could also be subject to UPF1 regulation. Perhaps one general function of the UPF1 pathway is to ensure that aberrant proteins do not accumulate in those instances in which regulated splicing (Baker, 1989) or incomplete RNA editing (Simpson and Shaw, 1989; Stuart, 1991) generates mRNAs lacking a functional open reading frame.

Regardless of its physiological role, identification of the upf1 loss-of-function mutations provides a valuable tool to study the nonsense-mediated mRNA decay pathway, cis-acting elements that promote nonsense-mediated mRNA decay can now be defined as sequences that accelerate mRNA decay in wild-type cells, but that are inactivated in strains deleted for the UPF1 gene.

As noted above, three other complementation groups of UPF mutants were identified in the same selection that identified upf1 (Culbertson et al., 1980). Since mutations in at least one of these genes, UPF3, also appear to reduce the rate of nonsense-mediated mRNA decay (Leeds et al., 1992), it is likely that yeast genetics will prove invaluable in the dissection of this mRNA degradation pathway.

A Inefficient Translation Termination and A-site mRNA Cleavage

Leif Isaksson and colleagues first discovered that the last two residues of the nascent chain influence translation termination (Mottagui-Tabar et al., 1994; Bjornsson et al., 1996). In general, C-terminal Pro, Asp, and Gly residues interfere with RF activity and promote stop-codon readthrough. These nascent peptide sequences also induce ssrA-tagging of full-length proteins in E. coli, with C-terminal Asp-Pro and Pro-Pro sequences leading to particularly high levels of tagging (Roche and Sauer, 2001; Hayes et al., 2002a). Ribosome-bound peptidyl prolyl-tRNA reacts with puromycin and aminoacyl-tRNA more slowly than other peptidyl-tRNA species (Muto and Ito, 2008; Wohlgemuth et al., 2008), perhaps accounting for the alternative reactions that occur during termination in vivo. Nascent peptide-induced ribosome pausing is associated with cleavage of the A-site stop-codon (Hayes and Sauer, 2003; Sunohara et al., 2004b). This RNase activity requires the ribosome and only occurs in response to translational pausing (Hayes and Sauer, 2003; Janssen and Hayes, 2009). A-site cleavage products accumulate to high levels in ΔssrA cells, but are difficult to detect in ssrA+ cells. Presumably, tmRNA activity facilitates the turnover of A-site truncated mRNA by releasing stalled ribosomes from the 3′-ends of these transcripts (Yamamoto et al., 2003). Together, these observations suggest that A-site mRNA cleavage provides a mechanism for tmRNA recruitment when ribosomes pause on full-length transcripts. A-site mRNA cleavage activity is very similar to that of ribosome-dependent mRNA interferases like RelE and YoeB. However, A-site cleavage still occurs in cells lacking the RelE, MazF, ChpBK, YoeB, YafQ, and YhaV toxins (Hayes and Sauer, 2003; Garza-Sánchez et al., 2008). Because the known mRNA interferases are not involved, we and Hiroji Aiba's group proposed that the ribosome itself may catalyze this A-site cleavage reaction (Hayes and Sauer, 2003; Sunohara et al., 2004a).

Subsequent work has shown that A-site mRNA cleavage is a more complicated process than originally appreciated. A-site cleavage does not occur in Δrnb mutants, which lack RNase II (Garza-Sánchez et al., 2009). RNase II is the major 3′-to-5′ exoribonuclease responsible for mRNA turnover in E. coli (Deutscher and Reuven, 1991). In Δrnb cells, prolonged translational pausing produces transcripts that are truncated + 12 nucleotides downstream of the A-site codon. This position corresponds to the ribosome leading edge or “toeprint” (Yusupova et al., 2001), and suggests that downstream mRNA is degraded to the 3′-border of the stalled ribosome. The effect of the Δrnb mutation on A-site mRNA cleavage is indirect because RNase II itself is unable to degrade mRNA into the ribosomal A-site. Purified RNase II only degrades mRNA to the + 18 position with respect to the A-site codon within the stalled ribosome (Garza-Sánchez et al., 2009). There are at least two models to explain these findings. First, deletion of rnb could alter the expression of other RNases such that + 12 cleavage is favored over A-site mRNA cleavage. This model is consistent with microarray data showing that global transcription is significantly altered in E. coli Δrnb mutants (Mohanty and Kushner, 2003). Alternatively, RNase II-mediated degradation of downstream mRNA could be a precondition for further degradation into the A-site codon, perhaps by facilitating the activity of another RNase.

1 Introduction

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that targets mRNAs undergoing premature translation termination for rapid degradation (He & Jacobson, 2015b; Kervestin & Jacobson, 2012; Lykke-Andersen & Bennett, 2014). The pathway was initially uncovered in Saccharomyces cerevisiae and Caenorhabditis elegans (Leeds, Peltz, Jacobson, & Culbertson, 1991; Peltz, Brown, & Jacobson, 1993; Pulak & Anderson, 1993) and later shown to be conserved from yeast to humans (Behm-Ansmant et al., 2007; Schoenberg & Maquat, 2012). NMD's function was originally thought to be limited to quality control, i.e., the elimination of mRNAs derived from genes harboring nonsense mutations to prevent the accumulation of potentially deleterious truncated polypeptides (He, Peltz, Donahue, Rosbash, & Jacobson, 1993; Pulak & Anderson, 1993). However, NMD also targets a significant fraction of apparently normal and physiologically functional wild-type mRNAs (Celik, Baker, He, & Jacobson, 2017; Schweingruber, Rufener, Zund, Yamashita, & Muhlemann, 2013), indicating that it also serves as a fundamental posttranscriptional regulatory mechanism for eukaryotic gene expression.

In all organisms examined the activation of NMD requires a set of conserved core regulatory factors, Upf1, Upf2, and Upf3 (He & Jacobson, 2015b; Kervestin & Jacobson, 2012). These three proteins interact with each other, the ribosome, and multiple translation and mRNA decay factors (Kervestin & Jacobson, 2012). Based on these molecular interactions, several potential functions have been proposed for the Upf factors, including remodeling terminating mRNPs (Franks, Singh, & Lykke-Andersen, 2010), releasing and recycling ribosomal subunits (Ghosh, Ganesan, Amrani, & Jacobson, 2010), and recruiting mRNA decay factors (He & Jacobson, 2015a; Nicholson, Josi, Kurosawa, Yamashita, & Muhlemann, 2014; Okada-Katsuhata et al., 2012). However, the exact roles for the Upfs, and their modes of action in NMD, remain largely unknown.

Transcripts targeted by NMD have been investigated using genome-wide high-density DNA microarrays over the last two decades. Depending on the organism or cell type, NMD usually targets about 5%–20% of the transcripts in a typical transcriptome (He et al., 2003; Lelivelt & Culbertson, 1999; Mendell, Sharifi, Meyers, Martinez-Murillo, & Dietz, 2004; Ramani et al., 2009; Rehwinkel, Letunic, Raes, Bork, & Izaurralde, 2005; Weischenfeldt et al., 2008) and these transcripts can be classified into several general categories. One category, exemplifying typical NMD substrates, includes mRNAs with a destabilizing premature termination codon (PTC) in their coding region. These transcripts are generated from endogenous genes harboring nonsense or frameshift mutations (He et al., 2003), pseudogenes (He et al., 2003; McGlincy & Smith, 2008), nonproductively rearranged genetic loci (Li & Wilkinson, 1998), or from alternative splicing events that lead to intron retention or inclusion of a PTC-containing exon (Jaillon et al., 2008; Lareau, Inada, Green, Wengrod, & Brenner, 2007; Lykke-Andersen et al., 2014; Ni et al., 2007). A second category contains mRNA-like transcripts with limited or no apparent coding potential, such as long noncoding RNAs (Kurihara et al., 2009; Lykke-Andersen et al., 2014; Tani, Torimura, & Akimitsu, 2013), small RNAs derived from intragenic regions (Smith et al., 2014; Thompson & Parker, 2007), or transcripts of inactivated transposable elements (He et al., 2003). A third category contains a subset of physiologically relevant transcripts that appear to be “normal,” such as mRNAs with upstream open reading frames (uORFs) (Arribere & Gilbert, 2013; Gaba, Jacobson, & Sachs, 2005; He et al., 2003), or with atypically long 3′-UTRs (Kebaara & Atkin, 2009; Singh, Rebbapragada, & Lykke-Andersen, 2008), or normal-looking wild-type mRNAs with no atypical features (He et al., 2003).

High-density DNA microarrays have limited dynamic ranges and are also unable to distinguish different transcript isoforms originated from the same genetic locus or homologous loci. To generate a comprehensive and high-resolution catalog of NMD-regulated transcripts, and to delineate the defining features of these transcripts in NMD targeting, we recently utilized RNA-Seq to reevaluate the effects of deleting the UPF1, UPF2, or UPF3 genes on the genome-wide expression of annotated yeast genes. Our new analyses confirm the previous results of microarray studies, but also uncover hundreds of new NMD-regulated transcripts that had escaped previous detection, including many intron-containing pre-mRNAs. Here, we describe the detailed experimental methods and the bioinformatics pipeline of our RNA-Seq experiments for analyzing the endogenous NMD substrates in yeast cells. We present detailed procedures for RNA isolation and construction of RNA-Seq libraries from wild-type and upf1 Δ, upf2 Δ, or upf3 Δ cells, and also outline the software used in our differential gene expression analyses.

What is the function of the protein release factor in translation quizlet?

Are release factors used during translation?

What is the function of release factors RF in prokaryotes?

What is the function of release factor RF1 or RF2 or RF3 in translation termination?